Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: TREM1-expression in the glioblastoma microenvironment is associated with poor patient outcomes. (A) Kaplan–Meier overall survival curves ( x -axis represents the time since glioma diagnostic) according to the tracks “high TREM1 expression, n = 270” versus “low TREM1 expression, n = 271” (median cut-off mode) are exhibited statistically significant differences ( P = 2.0 × 10 −10 ). (B) The graph illustrates an enhancement of the TREM1/ACTB mRNA ratios with the increase of the glioma grade. Results are shown as mean ± SD; the Student t -test determined significance; the difference is statistically significant between all glioma grades ( P < .05). Data from the Madhavan study for brain tumors and the Harris study for normal brains were obtained from the R2: platform; normal brain ( n = 44); grades I–II ( n = 100); grade III ( n = 85); grade IV ( n = 143). (C) TREM1 immunostaining in normal and glioma tissues (HPA005563 anti-TREM1 antibody, MiliporeSigma, Human Protein Atlas database). (D) Single-cell TREM1 RNA-seq data from adult and pediatric glioblastomas, GEO: GSE131928. (E) TREM1 expression in myeloid-derived macrophages from primary and recurrent glioblastomas (Immune glioblastoma Atlas database).

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Expressing, Diagnostic Assay, Immunostaining, RNA Sequencing, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: TREM1 expression is enriched in the glioblastoma peri-necrotic zones and in the glioblastoma/myeloid-derived cell hybrids. (A) Spatial RNA-seq signature of the myeloid-derived biomarkers and cytokines for glioblastomas of different molecular subtypes: classical, neural, proneural, mesenchymal, mesenchymal/neural, classical/neural, classical/mesenchymal, neural/proneural (36 samples total from 19 males and 17 females, Ivy Glioblastoma Project). The top two plots represent log2 of transcript expression; the bottom plot illustrates z -score. Note that the TREM1 is enriched in the peri-necrotic zones of glioblastomas of all molecular subtypes. (B) Western blot illustrates TREM1 and HBGB1 protein levels in normal and glioma samples of different grades; Actin is shown to confirm equal protein loading, TREM1/Actin ratios were calculated by using Image J program and are shown for each sample. (C) Chart illustrates expression of the myeloid cell markers in PDX cell lines of different subtypes (classical PDX1, PDX2; neural PDX3, PDX4; and mesenchymal PDX5; RNA-seq data). (D) Representative plots illustrate DP (double-positive, EGFP + RFP + ) cell selection by flow cytometry technique from dissociated tumor tissue from the immunocompetent glioblastoma mouse models; TRP-Fluc-EGFP and GL261-EGFP cells were intracranially injected in the immunocompetent mice with a wide-spread expression of RFP; DP cells were isolated 24 days after tumor implantation (see method). The representative image illustrates the bioluminescence tumor-reporter signal from mice injected with TRP-Fluc-EGFP cells. (E) Images illustrate representative TREM1 immunostaining in brain slices from glioblastoma mouse model with host cells expressing RFP and GL261-EGFP tumor; scale bar is 200 µm; the insert illustrates TREM1 expression on the host cell, which is undergoing fusion with tumor-cell in peri-necrotic zone. (F) Images illustrate representative TREM1 immunostaining in cultured DP (EGFP + RFP + ) cells isolated from the immunocompetent glioblastoma model with a wide-spread expression of RFP in host cells and GL261-EGFP tumor. Multi nuclei formations were observed in around 90% of DP cells. The scale bar is 200 µm. (G) Graph illustrates the percentages of the TREM1 + -cells in DP (EGFP + RFP + ) glioblastoma/host hybrids versus control GL261-EGFP cells. Results are shown as mean ± SD; the difference is statistically significant ( P < .005, Student t- test). At least five visual fields were analyzed for each analyzed slice, or sample of DP cells per mouse.

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Expressing, Derivative Assay, RNA Sequencing, Western Blot, Selection, Flow Cytometry, Injection, Isolation, Tumor Implantation, Immunostaining, Cell Culture, Control

Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: TREM1 + -myeloid-derived glioblastoma microenvironment is downregulated by an inhibitor of HuR dimerization in immunocompetent glioblastoma mouse models. (A, F) Graphs illustrate the weights of TREM1 + -myeloid-derived microenvironment in the dissociated brain tissue from immunocompetent TRP and GL261 mouse glioblastoma models at different treatment modalities versus normal (not tumor harboring) mice. Results are shown as mean ± SD; the statistically significant differences are marked with asterisks ( P < .05, Student t- test). The percentages of TREM1 + -myeloid-derived cells were the following in the experiment with TRP glioblastoma model: 1.2 ± 0.3%, n = 3 in normal mice without intracranial tumor injection, 7.7 ± 5.0%, n = 6 in mice injected with TRP tumor cells and treated with control vehicles, 1.5 ± 0.8%, n = 6 in mice intracranially injected with TRP tumor cells and treated with SRI42127. The percentages of TREM1 + -myeloid-derived cells were the following in the experiment with GL261 glioblastoma model: 2.8 ± 1.6%, n = 3 in normal mice without intracranial tumor injection, 7.0 ± 1.2%, n = 5 in mice intracranially injected with GL261 tumor cells and treated with control vehicles, 3.3 ± 1.5%, n = 5 in mice injected with GL261 cells and treated with SRI42127. (B, G) Representative plots illustrate flow cytometric profiling of brain myeloid-derived microenvironment from TRP and GL261 immunocompetent glioblastoma mouse models 20 days post intracranial tumor implantation. Plots illustrate representative examples of TREM1 expression in different subsets of myeloid-derived cells isolated from each group of mice (plots of each set represent data from the same mouse). (C, H) Graphs illustrate the weights of PMN and Mϕ cell subsets in brain tissue from TRP(C) and GL261(H) glioblastoma models at different treatment modalities. Results are shown as mean ± SD; the statistically significant differences are marked with asterisks ( P < .05, Student t- test). The percentages of tumor-infiltrated PMN and Mϕ were the following in the TRP-glioblastoma model: 5.7 ± 4.4%, n = 6 and 24 ± 22%, n = 6 after control-vehicle treatment versus 1.5 ± 0.9%, n = 6 and 20 ± 14%, n = 6 after SRI42127 compound treatment. The percentages of tumor-infiltrated PMN and Mϕ were the following in the GL261-glioblastoma model: 23 ± 9%, n = 5 and 7 ± 3%, n = 5 after control-vehicle treatment versus 13 ± 6, n = 5 and 1.9 ± 1, n = 5 after SRI42127 compound treatment. (D, I) Representative plots illustrate flow cytometric profiling of myeloid-derived tumor microenvironment in TRP and GL261 glioblastoma models treated with SRI42127 compound. Plots illustrate representative examples of TREM1 expression in different subsets of myeloid-derived cells isolated from each group of mice (plots inside of each set represent data from the same mouse). (E) Images of bioluminescence tumor-reporter signals from TRP glioblastoma model after treatment with SRI42127 compound (10 mice) versus control treatment with vehicles (10 mice) twenty days post intracranial tumor implantation. Note the robust tumor-reporter signal from 70% of mice in the control group versus 30% of mice in the group treated with SRI42127, suggesting the delay of the tumor progression in the mouse group treated with SRI42127.

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Derivative Assay, Injection, Control, Intracranial Tumor Implantation, Expressing, Isolation

Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: Glioblastoma/host cell fusion events are significantly reduced by SRI42127 in the immunocompetent GL261 glioblastoma model. (A) Representative plots (top) of DP (EGFP + RFP + ) glioblastoma/host cell hybrids selected by flow cytometry technique from brain tissue of GL261 mouse glioblastoma model treated with SRI42127 (T-SRI42127) versus control-vehicle treatment (T-Vh). Bottom plots illustrate corresponding EGFP + RFP +/− cell selection. GL261-EGFP cells (0.8 × 10 6 ) were injected in ten mice with wide-spread expression of RFP; mice were randomly divided into two equal groups and treated for 18 days starting from day four with SRI42127 (15 mg/kg, twice per day) or control vehicle. (B) The graph represents (EGFP + RFP + )/(EGFP + RFP − ) cell ratio in the mouse group treated with SRI42127 versus the control group. Results are shown as mean ± SD; the difference is statistically significant ( P < .05, Student t- test).

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Flow Cytometry, Control, Selection, Injection, Expressing

Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: Chemotaxis of myeloid-derived cells towards glioblastoma cells was attenuated by HuR and TREM1 inhibitors at the mimic hypoxic condition in vitro. (A) Representative images of migration of neutrophils and Mϕ labeled with red-fluorescence dye towards glioblastoma GL261-EGFP cells in the trans-well migration assay. Scale bars are 400 um in all images. (B, C) The graphs represent the areas (in percentages) covered by migrated neutrophils and Mϕ on the middle filter and at the bottom level in the trans-well migration assays towards glioblastoma GL261 and U87 cells plated at the bottom level 24 h prior to neutrophil and Mϕ placement in the middle chamber. Control experiments were performed without tumor cells on the bottom level. Results are shown as mean ± SD. (D, E) Graphs represent normalized migration of neutrophils and Mϕ towards glioblastoma cells in trans-well migration assay at different treatment modalities. The normalized values of neutrophil migration (detected on the bottom level) were 0.38 ± 0.13, n = 7 and 0.37 ± 0.06, n = 4 in the presence of LP17 (35 ng/ml) peptide towards GL261 and U87 cells, respectively, versus control-peptide treatments; and 0.13 ± 0.07, n = 7 and 0.17 ± 0.05, n = 7 in the presence of SRI42127 compound (5 µM) towards GL261 and U87 cells, respectively, versus control-vehicle treatments. The differences were significant for all conditions ( P < .05, Student t- test). The normalized values of Mϕ migration (detected in the middle filter) were 0.71 ± 0.09, n = 7 and 0.75 ± 0.12, n = 4 in the presence of LP17 (35 ng/ml) peptide towards GL261 and U87 cells, respectively, versus control-peptide treatments; and 0.70 ± 0.11, n = 7 and 0.75 ± 0.11, n = 5 in the presence of SRI42127 (5 µM) towards GL261 and U87 cells, respectively, versus control-vehicle treatments. The differences were significant for all conditions ( P < .05, Student t -test). The images on the right side of the graphs illustrate representative examples of myeloid-derived cell migration under different treatment conditions; scale bars are 400 µm; inserts framed with yellow represent images at high magnification.

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Chemotaxis Assay, Derivative Assay, In Vitro, Migration, Labeling, Fluorescence, Control

Journal: Neuro-Oncology Advances

Article Title: Targeting the TREM1-positive myeloid microenvironment in glioblastoma

doi: 10.1093/noajnl/vdac149

Figure Lengend Snippet: Cell fusion events between glioblastoma and myeloid-derived cells were significantly attenuated by HuR and TREM1 inhibitors at the mimic hypoxic condition in vitro. (A) Representative images of cell fusion events between myeloid-derived cells labeled with red-fluorescence dye and adherent glioblastoma GL261-EGFP cells at control (vehicle treatment) and SRI42127 (5 µM) treatment conditions. Scale bars are 400 µm. (B) Schematic illustration of cell fusion experiment between myeloid-derived cells labeled with red-fluorescence dye and tumor neurospheres expressing EGFP (top panel); representative images of cell fusion events between myeloid-derived cells labeled with red-fluorescence dye and PDX (XD456-EGFP) tumor neurospheres at 3D and single-cell levels (bottom panel). Scale bars are 400 µm. Yellow framed inserts represent images of neurospheres at high magnification. The graphs represent ratios of DP (EGFP + RFP + ) to (EGFP + + EGFP + RFP + ) cells in cell fusion assays between myeloid-derived cells and tumor neurospheres at different treatment conditions: SRI42127 treatment versus control (vehicle treatment) and LP17 treatment versus control scrambled peptide treatment. The ratios were the following: 0.49 ± 0.04, n = 6 (Vh-tr), 0.32 ± 0.03, n = 6 (SRI42127-tr) and 0.13 ± 0.03, n = 4 (Vh-tr), 0.09 ± 0.03, n = 4 (SRI42127-tr) in experiments with XD456 neurospheres and neutrophils or Mϕ, respectively; 0.52 ± 0.03, n = 4 (Vh-tr), 0.25 ± 0.03, n = 4 (SRI42127-tr) and 0.16 ± 0.02 n = 4 (Vh-tr), 0.10 ± 0.03, n = 4 (SRI42127-tr) in experiments with GL261 neurospheres and neutrophils or Mϕ, respectively; 0.45 ± 0.04, n = 6 (control-peptide), 0.23 ± 0.09, n = 6 (LP17) and 0.18 ± 0.03, n = 6 (control-peptide), 0.11 ± 0.03, n = 6 (LP17) in experiments with XD456 neurospheres and neutrophils or Mϕ, respectively; 0.49 ± 0.04, n = 5 (control-peptide), 0.29 ± 0.07, n = 5 (LP17) and 0.11 ± 0.03, n = 5 (control-peptide), 0.06 ± 0.02, n = 5 (LP17) in experiments with GL261 neurospheres and neutrophils or Mϕ, respectively; Results are shown as mean ± SD; the statistically significant differences are marked with asterisks ( P < .05, Student t- test). (C) The graphs represent normalized transcript expression in five PDX neurosphere cell lines after treatment with SRI42127 compound versus control vehicle treatment. Results were obtained by using RNA-seq analysis and are shown as mean ± SD; the statistically significant differences are marked with asterisks ( P < .05, Student t- test). (D) The graphs represent normalized tumor neurosphere proliferation in the presence of myeloid-derived cells at different treatment conditions: SRI42127 treatment versus control (vehicle treatment) and LP17 treatment versus control scrambled peptide treatment. The tumor-cell proliferation was determined by EGFP tumor-cell reporter signal. Results are shown as mean ± SD; the statistically significant differences are marked with asterisks ( P < .05, Student t- test). The proliferation was significantly declined in the range of 14–18% and 14–48% in the presence of LP17 and SRI42127 treatments, respectively. (E) Representative western blot illustrates TREM1 expression in the U87 cell line at different treatment conditions. GAPDH is shown for equal protein loading.

Article Snippet: Analysis of the TREM1 immunostaining in glioblastomas and normal brain tissues obtained from the Human Protein Atlas ( http://www.proteinatlas.org ) confirmed TREM1 overexpression in the glioblastoma microenvironment, particularly in peri-necrotic zones ( ).

Techniques: Derivative Assay, In Vitro, Labeling, Fluorescence, Control, Expressing, RNA Sequencing, Western Blot

Journal: Biomolecules

Article Title: Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration

doi: 10.3390/biom12081081

Figure Lengend Snippet: Overexpression of LGR4 in COAD tumor compared to normal tissue. ( A ) Genetic status of LGR4 in COAD patients obtained from the TCGA database. ( B ) Cancer types depended on gene alteration frequency obtained from TCGA database. ( C ) Expression levels of LGR4 genes in COAD patients were compared to normal colon tissue, and difference was conducted using LIMMA method (*, p < 0.05). ( D ) Expression profiles of LGR4 protein in normal colon tissue and COAD tissue were obtained from HPA database.

Article Snippet: Protein expression levels of LGR4 in normal tissues and COAD tissues were obtained from the Human Protein Atlas (HPA, https://www.proteinatlas.org/ , accessed on 11 November 2021).

Techniques: Over Expression, Expressing

Journal: Biomolecules

Article Title: Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration

doi: 10.3390/biom12081081

Figure Lengend Snippet: Kaplan–Meier analysis of COAD patients depended on LGR4 expression. Cut-off value is median value of LGR4 mRNA expression level. LGR4–1 is attributed to low expression of LGR4 in the COAD patients, and LGR4–2 is attributed to high expression of LGR4 in COAD patients. The curve comparison with the log-rank (Mantel-Cox) test revealed statistically significant differences as shown on graph ( p = 0.0021).

Article Snippet: Protein expression levels of LGR4 in normal tissues and COAD tissues were obtained from the Human Protein Atlas (HPA, https://www.proteinatlas.org/ , accessed on 11 November 2021).

Techniques: Expressing, Comparison

Journal: Biomolecules

Article Title: Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration

doi: 10.3390/biom12081081

Figure Lengend Snippet: Expression profile of identified genes in LGR4 -low expression group and LGR4 -high expression. ( A ) Volcano plot of genes in LGR4 -high expression group compared to LGR4 -low expression group. Threshold value of differentially expressed gene is |log 2 FoldChange| > 1.0 & p -value < 0.001. ( B ) Heatmap of differentially expressed genes identified in LGR4 -high expression group compared LGR4-low expression group.

Article Snippet: Protein expression levels of LGR4 in normal tissues and COAD tissues were obtained from the Human Protein Atlas (HPA, https://www.proteinatlas.org/ , accessed on 11 November 2021).

Techniques: Expressing

Journal: Biomolecules

Article Title: Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration

doi: 10.3390/biom12081081

Figure Lengend Snippet: ( A ) Protein–protein interaction network of DEGs identified in LGR4 -high expression group compared to LGR4 -low expression group. The red circle-labeled hub genes of DEGs are ALB , F2 , APOA2 , CYP1A1 , SPRR2B , APOA1 , APOB , CYP3A4 , SST , and GCG . ( B ) Heatmap of Pearson’s correlation matrix of hub genes in LGR4 -high expression group compared to LGR4 -low expression group. Threshold value identified as significant is p value < 0.05.

Article Snippet: Protein expression levels of LGR4 in normal tissues and COAD tissues were obtained from the Human Protein Atlas (HPA, https://www.proteinatlas.org/ , accessed on 11 November 2021).

Techniques: Expressing, Labeling

Journal: Biomolecules

Article Title: Bioinformatics Analysis of LGR4 in Colon Adenocarcinoma as Potential Diagnostic Biomarker, Therapeutic Target and Promoting Immune Cell Infiltration

doi: 10.3390/biom12081081

Figure Lengend Snippet: ( A ) Unsupervised clustering analysis of COAD based on ssGSEA score of 24 immune cell types. P001 is attributed to COAD patients with lower expression level of LGR4 , and P002 is attributed to COAD patients with higher expression level of LGR4 . ( B ). Correlation between LGR4 expression level and infiltration level of major immune cells.

Article Snippet: Protein expression levels of LGR4 in normal tissues and COAD tissues were obtained from the Human Protein Atlas (HPA, https://www.proteinatlas.org/ , accessed on 11 November 2021).

Techniques: Expressing