Journal: Oncotarget

Article Title: Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

doi:

Figure Lengend Snippet: A. P62 mRNA expression in human cancers compared to corresponding normal tissues. The graph was derived from microarray data of Oncomine. B-D. Expression of oncogenes leads to p62 accumulation. MCF10A human mammary epithelial cells were transduced with retroviruses (B,C) or lentiviruses (D) expressing the corresponding oncogenes, and (after brief selection) levels of p62 were assessed by immunoblotting with anti-p62 antibody. Duplicates (separate infections) are shown in B and D, and effect of increased levels of RAS virus in C. Anti-actin antibody was used as a loading control.

Article Snippet: MCF10A were from ATCC; MCF10A cells expressing PIK3CA were kindly provided by Dr. T. Waldmann.

Techniques: Expressing, Derivative Assay, Microarray, Transduction, Selection, Western Blot, Virus, Control

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Oxaliplatin-resistant SW620, SW480, HCT116, and HT29 colon cancer cell lines demonstrate similar or enhanced sensitivity to TRAIL compared to their parental counterparts after 24 hr of treatment. N = 3 (biological replicates); n = 9 (technical replicates). ( B ) IC50 values were calculated using a variable slope four-parameter nonlinear regression. ( C ) Representative Annexin-V/PI flow plots comparing SW620 parental and OxR cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. The four quadrants represent viable cells (bottom left), early apoptosis (bottom right), necrosis (top left), and late apoptosis (top right). ( D ) Representative flow plots of JC-1 assay after treatment with 1000 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. ( E ) Mitochondrial depolarization as a function of TRAIL concentration for SW620 parental and OxR cell lines. N = 3 (n = 9). For all graphs, data are presented as mean ± SD. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). Figure 1—source data 1. Raw viable cell counts from Annexin-V/PI assays and percent depolarized mitochondria in SW620 cells (panels A and E).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) SW620, SW480, HCT116, and HT29 cells were treated with various concentrations of oxaliplatin for 72 hr and cell viability was measured using an MTT assay. IC50 values were calculated using a variable slope four-parameter nonlinear regression. Data are presented as mean ± SEM. N = 2 (n = 12). ( B ) Counts of successfully invasive cells after a 4-day Transwell assay with an initial seeding of 200,000 cells. N = 2 (n = 6). * p<0.05; **p<0.01 (unpaired two-tailed t-test). Figure 1—figure supplement 1—source data 1. Raw data from MTT cell viability assays after oxaliplatin treatment (panel A). Figure 1—figure supplement 1—source data 2. Invasive cell counts from Transwell assays (panel B).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: MTT Assay, Transwell Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Sensitization of oxaliplatin-resistant SW620, HCT116, HT29, and SW480 cell lines compared to their parental counterparts as a function of TRAIL concentration. ( B ) Maximum TRAIL sensitization for each cell line between the tested concentrations of 0.1–1000 ng/ml. Data are presented as mean ± SEM. Figure 1—figure supplement 2—source data 1. TRAIL sensitization calculations.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Concentration Assay

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Representative flow plots of JC-1 assay after treatment with 200 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. ( B ) Mitochondrial depolarization as a function of TRAIL concentration for HCT116 parental and OxR cell lines. Data are presented as mean ± SD. N = 3 (n = 9). ****p<0.0001 (unpaired two-tailed t-test). Figure 1—figure supplement 3—source data 1. Percent depolarized mitochondria in HCT116 cells measured from JC-1 assays.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, B ) Volcano plots of RT-PCR Apoptosis Profiler arrays demonstrate downregulation of CASP10 in OxR phenotypes. N = 3. ( C ) CRISPR/Cas9 knockout of caspase-10 in SW620 parental cells was confirmed via western blot. sgRNA/Cas9 ribonucleoprotein complexes reduced caspase-10 expression by 93% compared to cells treated with Cas9 alone. ( D ) CASP10 knock-out (KO) cells demonstrate slight decreases in viability when treated with TRAIL compared to Cas9 control. Data are presented as mean ± SD. N = 3 (n = 9). ( E ) Representative Annexin-V/PI flow plots comparing SW620 parental (Cas9 only) and CASP10 KO cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. ( F ) Depletion of caspase-10 did not have a significant effect on TRAIL sensitization (unpaired two-tailed t-test). Data are presented as mean + SEM. N = 3 (n = 9). Figure 2—source data 1. Apoptosis microarray data in HCT116 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel A). Figure 2—source data 2. Apoptosis microarray data in SW620 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel B). Figure 2—source data 3. Western blot images (raw and annotated) confirming CASP10 KO (panel C). Figure 2—source data 4. Quantification of CASP10 KO from western blots (panel C). Figure 2—source data 5. Cell viability and TRAIL sensitization calculations in CASP10 KO cells (panels D and F).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Reverse Transcription Polymerase Chain Reaction, CRISPR, Knock-Out, Western Blot, Expressing, Control, Two Tailed Test, Microarray, Generated

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, B ) Confocal micrographs of HCT116 and SW620 cells, respectively. Red channel represents DR4, green is lipid rafts, and blue is DAPI (nuclei). Scale bar = 30 μm. ( C ) Quantification of DR4 area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. Data are presented as mean + SEM from N = 3 independent experiments. ***p<0.001; ****p<0.0001 (unpaired two-tailed t-test). ( D ) OxR cells had increased surface expression of DR4 in non-permeabilized cells analyzed via flow cytometry. # Significant according to a chi-squared test (see ). ( E ) Western blots for DR4 in whole cell lysates of parental and OxR cells. ( F ) Quantification of western blots from three independent experiments (N = 3). Data are presented as mean + SEM. *p<0.05 (unpaired two-tailed t-test). ( G ) Percentage of apoptotic SW620 cells after treatment with 0.01–10 µg/ml mapatumumab (sum of early and late-stage apoptotic cells from Annexin/PI staining). Data are presented as mean ± SD. N = 3 (n = 6). ****p<0.0001 (multiple unpaired two-tailed t-tests). ( H ) Cell viability of SW620 cells after mapatumumab treatment, determined by Annexin-V/PI staining. Data are presented as mean ± SD. N = 3 (n = 6). ( I ) Maximum mapatumumab sensitization within OxR cell lines compared to their parental counterparts. Data are presented as mean + SEM. Figure 3—source data 1. Quantification of DR4 area per cell in HCT116 and SW620 cells (panel C). Figure 3—source data 2. Western blot images (raw and annotated) for DR4 (panel E). Figure 3—source data 3. Quantification of DR4 from western blots (panel F). Figure 3—source data 4. Cell viabilty and percent apoptosis in SW620 cells after mapatumumab treatment (panels G-I).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Expressing, Flow Cytometry, Western Blot, Staining

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A–D ) Confocal micrographs and DR5 quantification of HCT116, SW620, SW480, and HT29 cells, respectively. Red channel is death receptor 5, green is lipid rafts, and blue is DAPI (nuclei). Scale bar = 30 μm. ** p<0.01; ****p<0.0001 (unpaired two-tailed t-test). Data are presented as mean + SEM. For each cell line, N = 75 cells were analyzed. ( E ) Oxaliplatin-resistant (OxR) cells only demonstrate increased surface expression of DR5 in non-permeabilized SW620 cells. # Significant according to a chi-squared test (see ). Figure 3—figure supplement 4—source data 1. Quantification of DR5 area per cell for all cell lines.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Expressing

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Cell viability of HCT116 cells after 0.01–10 µg/ml mapatumumab treatment, determined by Annexin-V/PI staining. ( B ) Percentage of apoptotic SW620 cells after mapatumumab treatment (sum of early and late-stage apoptotic cells from Annexin/PI staining). For all graphs, data are presented as mean + SD. N = 3 (n = 6). *p<0.05; ****p<0.0001 (multiple unpaired two-tailed t-tests). Figure 3—figure supplement 7—source data 1. Cell viabilty and percent apoptosis in HCT116 cells after mapatumumab treatment.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Staining, Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Composite images and binary projections of DR4/LR colocalization areas in HCT116 and SW620 cell lines. Lipid raft and DR4 binary images were generated for a specified threshold, then multiplied by one another to generate images with positive pixels in double-positive areas. Red is DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. ( B ) Quantification of DR4 and lipid raft colocalization area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). ( C ) Correlation between the fold change in DR4/LR colocalization (OxR phenotype/parental) and maximum TRAIL sensitization observed by the OxR phenotype for each of the four cell lines (simple linear regression analysis). ( D ) Lipid raft fractions were isolated and analyzed for DR4 via western blot in parental and OxR cells. ( E ) Quantification of lipid raft DR4 blots in ( D ). *p<0.05 (unpaired two-tailed t-test). For all graphs, data are presented as mean + SEM. ( F ) Förster resonance energy transfer (FRET) efficiencies of FITC-labeled DR4 (donor) and Alexa 555-labeled lipid rafts (acceptor) in parental and OxR cells analyzed via flow cytometry. **p<0.01; ***p<0.001 (unpaired two-tailed t-test). Figure 4—source data 1. Quantification of LR-colocalized DR4 area per cell in HCT116 and SW620 cells (panel B). Figure 4—source data 2. Correlation analysis of LR-colocalized DR4 area per cell with TRAIL sensitization (panel C). Figure 4—source data 3. Western blot images (raw and annotated) for DR4 from LR isolates (panel D). Figure 4—source data 4. Quantification of LR DR4 from western blots (panel E). Figure 4—source data 5. FRET efficency calculations (panel F).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Generated, Two Tailed Test, Isolation, Western Blot, Förster Resonance Energy Transfer, Labeling, Flow Cytometry

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A ) Quantification of DR5/LR colocalization in HCT116, SW620, SW480, and HT29 cells. *p<0.05; **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). For each cell line, N = 75 cells were analyzed. ( B ) Correlation of total DR5 area per cell and ( C ) DR5/LR colocalization with maximum TRAIL sensitization observed in OxR cells (linear regression analysis). For all graphs, data are presented as mean + SEM. ( D ) Western blots show DR5 was undetectable in lipid raft isolated fractions. Figure 4—figure supplement 2—source data 1. Quantification of LR-colocalized DR5 area per cell (panel A). Figure 4—figure supplement 2—source data 2. Correlation analysis of DR5 and LR-colocalized DR5 area per cell with TRAIL sensitization (panels B and C). Figure 4—figure supplement 2—source data 3. Western blot images (raw and annotated) for DR5 from LR isolates (panel D).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test, Western Blot, Isolation

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: ( A, G ) SW620 oxaliplatin-resistant (OxR) and HCT116 OxR cells, respectively, treated for 24 hr with a combination of TRAIL and 5 µM nystatin. ( B, H ) SW620 OxR and HCT116 OxR cells, respectively, showed a significant decrease in TRAIL sensitization when treated in combination with nystatin. N = 3 (n = 9). ( C, I ) Treatment with 5 µM nystatin significantly decreased DR4/LR colocalization area in SW620 OxR and HCT116 OxR cells, respectively. For each cell line, N = 40 cells were analyzed. ( D, J ) SW620 Par and HCT116 Par cells, respectively, treated for 24 hr with a combination of TRAIL and 70 µM resveratrol. N = 3 (n = 9). ( E, K ) SW620 Par and HCT116 Par cells, respectively, showed a significant increase in TRAIL sensitization when treated in combination with resveratrol. N = 3 (n = 9). ( F, L ) Treatment with 70 µM nystatin significantly increased DR4/LR colocalization area in SW620 Par and HCT116 Par cells, respectively. For each cell line, N = 40 cells were analyzed. ( M ) Representative composite images and binary projections of DR4/LR colocalization in SW620 OxR cells before and after nystatin treatment. ( N ) Representative composite images and binary projections of DR4/LR colocalization in parental SW620 cells before and after resveratrol treatment. Red represents DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test for all graphs). ( A, D, G, J ) Data are presented as mean ± SD. ( B, C, E, F, H, I, K, L ) Data are presented as mean + SEM. Figure 5—source data 1. Cell viability after TRAIL combination treatments with resveratrol and nystatin (panels A, D, G, J). Figure 5—source data 2. TRAIL sensitization calculations after resveratrol and nystatin (panels B, E, H, K). Figure 5—source data 3. Quantification of LR-colocalized DR4 after resveratrol and nystatin treatment (panels C, F, I, L).

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet: For each cell line, N = 40 cells were analyzed. Data are presented as mean + SEM. *p<0.05 (unpaired two-tailed t-test). Figure 5—figure supplement 1—source data 1. Quantification of the effects of resveratrol and nystatin on DR5 colocalization with LRs in HCT116 and SW620 cells.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: Two Tailed Test

Journal: eLife

Article Title: Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

doi: 10.7554/eLife.67750

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HCT116 carcinoma, colorectal , ATCC , #CCL-247 , RRID: CVCL_0291 McCoy’s 5A Media.

Techniques: MTT Assay, Recombinant, Software, Membrane, Gene Knockout, Labeling, Control, Neutralization, Clinical Proteomics, Isolation, Magnetic Beads

Journal: Cell Reports Medicine

Article Title: Combinatorial targeting of glutamine metabolism and lysosomal-based lipid metabolism effectively suppresses glioblastoma

doi: 10.1016/j.xcrm.2024.101706

Figure Lengend Snippet:

Article Snippet: Authenticated (short tandem repeat profiling) human GBM cell lines, U251 (Male) and U373 (Male) from Sigma, U87 (HTB-14) (Female), T98 (CRL-1690) (Male), LN (CRL-2611) (Female) cells American Type Culture Collection (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% HyClone fetal bovine serum (FBS), and 4 mM glutamine.

Techniques: Plasmid Preparation, Virus, Microarray, Recombinant, Modification, Isolation, Clinical Proteomics, Acid Assay, Amplex Red Cholesterol Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Lysis, Protease Inhibitor, Luciferase, Transfection, cDNA Synthesis, Control, Software

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Lung squamous carcinoma is associated with inflammatory monocytes. a Kaplan–Meier plots of overall survival in lung squamous carcinoma (LUSC) patients ( n = 380) from The Cancer Genome Atlas (TCGA) categorized by mRNA subtype. b Overall survival comparing each mRNA subtype with all others. c Hierarchical clustering of ( n = 4291) genes expressed in 348 patients from the LUSC TCGA RNA-seq dataset. d Overall survival comparing Classical versus Secretory subtypes. e Network visualization of Ingenuity Pathway Analysis (IPA) of all differentially expressed genes in the upper portion of the heat map (shown in panel c ) that are statistically significant ( p < 0.05) in terms of patient survival (in the comparison high ( ≥ median) vs. low ( < median) gene expression), termed “survival genes”. (Gray: least significant, Red: most significant) f . Gene ontology analysis to investigate the biological processes most linked with genes differentially expressed, moving between the Classical and Secretory subtypes (see the Supplementary Methods for details). Individual bars represent most statistically significant GO terms in either the Classical (red bars) or Secretory (green bars) subtype. g Gene Set Enrichment Analyses (GSEA) of the LUSC TCGA dataset. The GSEA is performed going from Secretory to Classical; the GSEA ‘mountain plots’ show only the two most divergent subtypes. Gene set names were shortened to fit this figure. h Table showing 9 genes from the upper portion of the heat map that are associated with reduced overall survival and are markers of monocytes and macrophages. Genes with log-rank p≤0.001 are highlighted in red

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: RNA Sequencing, Comparison, Gene Expression

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Inflammatory monocytes associate with survival in LUSC. a Kaplan–Meier plots of overall survival in lung squamous carcinoma (LUSC) patients split by median (left, p < 0.0001) and quartile (right, p = 0.0006) expression levels of CD14. P -values are obtained with the log-rank test; FDR were calculated according to Benjamini and Hochberg. b Proportion of patients by mRNA subtype that have CD14 expression levels above (red) or below (black) the median CD14 expression level. Binomial tests for proportions were performed. (Black asterisks: significant enrichment below the median, red asterisks: Significant enrichment above the median). c Kaplan–Meier plots of overall survival in LUSC by expression levels of CCL2 ( p = 0.001), CCL3 ( p = 0.018) and CSF1 ( p = 0.015) expression. d Pearson’s correlations of CCL2, CCL3, and CSF1 chemokines versus CD14 (gene expression). e Dynamic range of mRNA expression of CD14 and CCL2 for each LUSC mRNA subtype. P -values were obtained with analysis of variance. The purple shading for CD14 expression represents samples in the ‘IM-rich subset’ (above the median CD14 expression level). f Dynamic range of mRNA expression of CCL3 and CSF1 by LUSC mRNA subtype. g Representative multiplex IHC for CD14 (green), CCR2 (red) and pan-cytokeratin (light blue) in a LUSC tumor sample, and enumeration of CD14+/CCR2+ cells in CK+ and CK- regions. #1-3 represent CK- regions, #4 represents a CK + region. Scale bar 100 μm. h Representative dual CD14+/CCR2+ cells (white arrows) in CK- (#1-3) and CK+ (#4) regions. Note: CK + region shown in white channel to more easily appreciate green and red. Scale bar 25 μm. i Enumeration of CD14+/CCR2+ cells in CK- and CK+ regions by mRNA subtype. Classical ( n = 14), Basal ( n = 9), Primitive ( n = 6) and Secretory ( n = 12). p -values were obtained with Student’s t-test in comparison to the Classical subtype. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Expressing, Gene Expression, Multiplex Assay, Comparison

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: IMs have the strongest correlation with CD14 and LUSC survival. a At top, the mRNA subtype of each sample is displayed (Classical: red, Basal: blue, Primitive: black, Secretory: green); CD14 expression levels sorting from left (lowest) to right (highest) for the corresponding samples are displayed below. Heat maps of the CD14 + populations that are associated with a statistically significant survival in LUSC are arranged by the most (IMs) to the least (MDSCs) statistically significant. CD14 is a marker both of IMs and MDSCs but is not shown in their heat maps, to avoid data redundancy. Note for the heat map figure legend: gray represents ‘null normalized values’ (NNV). b Scatter plot of overall survival results and CD14 scores for the 9 CD14 + immune cell types. Survival is represented with hazard ratio (HR) ± 95% confidence intervals. Values above 1 indicate worse survival based on cell type density. The CD14 score is the rank of the ratio between the average cell type density score of samples with high ( ≥ median) vs. low ( < median) CD14 levels. c Log-rank p -values of overall survival for the 9 CD14 + immune cell types (high vs. low cell density score). The red bars show statistically significant cases, while black bars are used when the statistical significance threshold of 0.05 is not reached

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Expressing, Marker

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: TNFα activation of NFκB promotes CCL2-mediated IM recruitment. a Microarray expression data (left) comparing murine bronchial epithelial cells (MBECs), parental KLN205 and the LN4K1 sub-clone. Top upstream pathways (right) from Ingenuity Pathway Analysis (IPA) are shown for the differentially regulated genes shown in brackets. b An upstream network visualization from IPA of all over-expressed genes (all nodes) in the upper portion of the heat map shown in Fig. with significant log-rank (survival analysis) p -value ( < 0.05). TNFα and NFκB (blue nodes) were amongst the top upstream regulators known to have direct roles (black lines) in promoting CCL2, CCL3 and CSF1 chemokines (the degree of redness corresponds with increasing statistical significance). c Relative expression of TNFα. d CCL2, CCL3 and CSF1 by qPCR. Data are averages ± s.e.m. P -values were obtained with Student’s t-test in comparison with KLN205. e Relative levels of CCL2 as measured by ELISA from secreted media of cells growing in vitro or, f , from plasma of tumor-bearing mice. Data are averages ± s.e.m. g Relative mRNA expression of CCL2 and p65 in LN4K1 cells following treatment with control or p65 siRNA with or without exogenous TNFα (100 ng/mL). h Relative expression of CCL2 mRNA (top) and phospho-p65 and p65 protein (bottom) following treatment with DMSO or an IKKβ inhibitor (Compound A, 5 μM) for 5 h. i Relative IM counts in the bone marrow, blood, spleen from healthy DBA2 mice versus those with LN4K1 tumors. j Relative IM, TAM and TReg counts from the lungs of healthy versus LN4K1-bearing mice. IMs were also assessed in age-matched DBA2 mice following HBSS ‘Mock’ injection. P -values obtained with one-sided Student’s t-test, n = 5 mice/group for f , i , and j . * P < 0.05, ** P ≤0.01, *** P ≤0.001

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Activation Assay, Microarray, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, In Vitro, Clinical Proteomics, Control, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: CCL2-mediated IM recruitment is critical for LUSC metastasis. a Relative expression of CCL2 for KLN205 and sub-clones. b Quantification of luciferase signal. c Representative images obtained 10 days after cell injection of (i) KLN205-Scr ORF, (ii) KLN205-CCL2 ORF, (iii) LN4K1-Cntrl shR, (iv) LN4K1-CCL2 shR#1 and (v) LN4K1-CCL2 shR#2. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. d Survival plots of mice following tail vein injection of KLN205 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. e Number of IMs per lung lobe, n = 12 lobes/group. f Survival plots of mice following tail vein injection of LN4K1 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. g Number of IMs per lung lobe, n = 12 lobes/group. h Schematic (left) and quantification of luciferase signal (right) of mice treated with vehicle or PF-04136309 to assess effects on established metastases. i FACS plots and ( j ) quantification of percent IMs in the blood and ( k ) right lung of LN4K1-bearing mice. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 5 mice/group. l , FACS analysis of percent immune infiltrates for TAMs (gated on F480), DCs (gated on SiglecF-/CD11c), CD4, CD8, Tregs (gated on TCRb + ) and NK cells (gated on SiglecF-/B220-/TCRb-). m Schematic (left) and quantification of luciferase signal (right) of mice treated at the time of cell injection to assess effects on preventing metastasis. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. n.s. = non-significant, * P ≤0.05, *** P < 0.001. For panel b , * FDR < 0.05, ** FDR < 0.01

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Expressing, Clone Assay, Luciferase, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Factor XIIIA in IMs promotes fibrin cross-linking and LUSC invasion. a Relative mRNA expression of F13a1 from sorted IMs and RMs, n = 3 mice. b Immunofluorescent (IF) imaging of IMs and RMs comparing FXIIIA (red) expression. c Dual staining for FXIIIA (red) and CD11b (green) in IMs. Contents of dotted white box are enlarged under each panel. d Confocal imaging of IMs for FXIIIA (red). White arrows point toward podosome-like structures. Scale bar: 5 μm; nuclei were stained with Hoechst (panels b – d ). e Fibrin cross-linking patterns by western blotting using a polyclonal anti-human fibrin(ogen) antibody using freshly sorted IMs and RMs (Low = 25k cells, High = 100k cells), with or without the FXIIIA-inhibitor, T101. f Percent invadopodia of LN4K1 cells growing in either unfractionated (UF Fgn, left) or Peak 1 (FXIIIA-depleted) fibrinogen (right). White arrows show evidence of invadopodia. Scale bar 50 μm. Data are averages ± s.e.m. P -values obtained with Student’s t-test. g Schematic of co-culture model (top). Representative images of LN4K1-GFP cells growing in either UF Fgn or Peak 1 with or without low (100k) or high (300k) IMs. FDR = 0.0001 for all statistical comparisons shown. Groups in e + g treated with the T101 were dosed at 50 μM. Scale bar 12.5 μm. Data are averages ± s.e.m. P -values were obtained with Student’s t-test. h Invaded LN4K1-GFP cells per high power field (HPF) at 24 h following seeding into either UF Fgn or Peak 1 Fgn alone or co-cultured with IMs with or without T101 (50 μM). FDR < 0.01 for all statistical comparisons shown. i Representative images (left) of LN4K1-GFP cells co-cultured with IMs from either wild-type or F13a1 −/− mice. Number of invadopodia positive LN4K1-GFP cells (right) per HPF at 24 h when growing in UF Fgn or Peak 1 Fgn alone or co-cultured with IMs from either wild-type or F13a1 −/− mice. Scale bar 12.5 μm. Data are averages ± s.e.m. P -values were obtained with Student’s t-test. FDR < 0.01 for all statistical comparisons shown. ** P < 0.01, *** P < 0.0001

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Expressing, Imaging, Staining, Western Blot, Co-Culture Assay, Cell Culture

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: FXIIIA and fibrin cross-linking is associated with LUSC progression. a Representative IHC images (left) of tumors from the LUSC tissue microarray with low or high cross-linked fibrin ( n = 96 patients). Kaplan-Meier plot of recurrent-free survival following surgical resection. P -values were obtained with the log-rank test. b Kaplan–Meier plot of overall survival in LUSC patients split by median expression levels of F13a1. P -values were obtained with the log-rank test. c Relative expression levels of F13a1 in THP1 monocytes stably expressing either F13 ORF or shRNAs against F13a1. P -values were obtained with Student’s t-test. d Schematic (left) and quantification of distant micro-metastases (right) following infusions of THP1 monocytes. NSG mice were injected via tail vein with LN4K1-OgNLuc cells on Day 1, followed by daily infusions of THP1 cells in the respective groups for Days 1–4. Mice were necropsied on Day 7, lungs were dissociated and FACS analysis performed for EpCAM + cells to quantify micro-metastases per lobe. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 3 mice/group. n.s. = non-significant, * P < 0.05, *** P < 0.001

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Microarray, Expressing, Stable Transfection, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Schematic of the ‘IM-rich subset’ of lung squamous carcinoma. TNFα activation of the canonical NFκB leads to LUSC cell secretion of chemo-attractant CCL2, which stimulates the bone marrow to release inflammatory monocytes (IMs) into circulation. The IMs bring large payloads of FXIIIA into the tumor microenvironment, leading to cross-linked fibrin, LUSC invadopodia formation and progression

Article Snippet: KLN205 lung squamous cell carcinoma cells were obtained from the ATCC, parental KAL cells were kindly provided by Dr. Yinling Hu (National Cancer Institute, Frederick, MD) and 344SQ lung adenocarcinoma cells were kindly provided by Dr. John Kurie (M.D.

Techniques: Activation Assay

Journal: Cancer cell

Article Title: Apoptotic cell-derived extracellular vesicles promote malignancy of glioblastoma via intercellular transfer of splicing factors

doi: 10.1016/j.ccell.2018.05.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HBEC5i , ATCC , Cat# CRL-3245.

Techniques: Virus, Microarray, Clinical Proteomics, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Plasmid Preparation, Labeling, Polymer, Membrane, Immunoprecipitation, Flow Cytometry, Bradford Protein Assay, RNA Sequencing, shRNA, Sequencing, Cloning, Software