human colorectal cancer cell line dld1  (Procell Inc)

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/TRMT61A‐mediated m 1 A modification drives cellular senescence in CRC. (A) Representative immunohistochemistry (IHC) images showing co‐expression patterns of m 1 A, p21, p16, TRMT6, and TRMT61A in CRC tumor tissues. The upper and lower rows displayed the two regions with highly and lowly concurrent staining of three markers, respectively. Scale bars, 100 µm. (B) Pearson's correlation heatmap that visualized the relationships between the IHC scores of intracellular markers, including TRMT6, TRMT61A, m 1 A, p21, and p16 in CRC tumor tissues (NEPDC cohort, n = 58) was shown. The numbers in the boxes represented Pearson correlation coefficients (r), and the color scale indicated the strength of the correlation. (C) The TRMT6/61A‐mediated RNA m 1 A modification depended on the catalytic domains. We constructed the DLD1 and HCT116 cells with stable overexpression of vector (CTRL), wild‐type TRMT6/61A (OE‐WT), and catalytically inactive mutant TRMT6‐R377L/TRMT61A‐D181A (OE‐MUT) (top), and the dot blot analysis showed global RNA m 1 A levels were elevated in OE‐WT cells rather than OE‐MUT cells compared with CTRL cells (bottom). The methylene blue (MB) staining served as a loading control for RNA. (D) Gene set enrichment analysis (GSEA) of differentially expressed genes between OE‐WT and CTRL groups of DLD1 cells based on their RNA‐seq profiles. The plot showed that cellular senescence was active in the cells highly expressing wild‐type TRMT6/61A. NES: Normalized enrichment score. (E) Western blot analysis demonstrating increased protein levels of senescence markers p21 and p16 in the OE‐WT rather than the OE‐MUT group of DLD1 and HCT116 cells compared with the CTRL group cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) Representative images of senescence‐associated β‐galactosidase (SA‐β‐Gal) staining in the three groups of DLD1 and HCT116 cells. The senescent cells were identified by their blue color. Scale bars, 100 µm. (G) Proportions of SA‐β‐Gal‐positive cells in the three groups of DLD1 and HCT116 cells from three independent experiments. The percentage of SA‐β‐Gal‐positive cells was determined by quantifying both positive (blue‐stained) and total cells from three randomly selected high‐power fields (100×) per well using ImageJ software. Data are presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Modification, Immunohistochemistry, Expressing, Staining, Construct, Over Expression, Plasmid Preparation, Mutagenesis, Dot Blot, Control, RNA Sequencing, Western Blot, Software

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/TRMT61A‐mediated m 1 A modification promotes cellular senescence by enhancing the translational efficiency of ARG2 (A) Schematic diagram illustrating the tRNA m 1 A‐seq workflow. (B) Scatter plot of differentially m 1 A‐modified tRNA between DLD1 cells with stable overexpression of vector (CTRL) and wild‐type TRMT6/61A (OE‐WT) based on their tRNA‐m 1 A‐seq profiles. Red dots represented tRNAs with significantly upregulated m 1 A methylation levels (Fold Change > 1 and p < 0.05) in the OE‐WT group. (C) Gene ontology (GO) enrichment analysis of transcripts with upregulated translation efficiency in OE‐WT cells compared to CTRL cells, as determined by ribosome profiling (Ribo‐seq). The enriched biological processes were ranked by p ‐value, and the positive regulation of cellular senescence involving ARG2 and YPEL3 was highlighted. (D) A heatmap illustrating the top 30 genes with the highest increase in translation efficiency in OE‐WT cells, and the senescence‐associated gene, ARG2 , was highlighted. (E) RT‐qPCR analysis of ARG2 and YPEL3 mRNA expression in the CTRL and OE‐WT cells. Data were presented as scatter dot plots showing individual data points from three independent experiments ( n = 3), along with mean ± SD. (F) Western blot analysis showed that the protein levels of ARG2 and phosphorylated mTOR (p‐mTOR) were increased in OE‐WT cells rather than OE‐MUT cells, compared with CTRL cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (G, H) Overexpression of ARG2 (OE‐ARG2) induced cellular senescence similar to wild‐type TRMT6/61A. (G) Western blot analysis confirmed ARG2 overexpression and mTOR activation. (H) SA‐β‐Gal staining images and quantification. The percentage of positive cells was quantified from three random fields. (I, J) Knockdown of ARG2 rescued TRMT6/61A‐induced senescence. The Western blot showed that ARG2 knockdown reversed the increased levels of p‐mTOR, p21, and p16 in OE‐WT HCT116 and DLD1 cells (I), and the decreased SA‐β‐Gal‐positive cells after ARG2 knockdown in OE‐WT cells with representative images were shown (J). (K) The mTOR pathway is required for TRMT6/61A‐induced senescence. Each group of cells was treated with the mTOR inhibitor rapamycin for 48 h. Representative images and quantification of SA‐β‐Gal staining showed that rapamycin treatment reversed the senescent phenotype in OE‐WT cells. Scale bars, 100 µm. (L) The dual‐luciferase reporter system. A schematic diagram of the reporter constructs is provided in Figure . We constructed reporter plasmids by inserting six‐repeated cognate codons for either Asp (GAC) or Glu (GAG)—decoded by the m 1 A‐hypermethylated tRNA‐Asp‐GTC and tRNA‐Glu‐CTC, respectively—immediately upstream of the firefly luciferase gene. The relative luciferase activity was measured. The enhanced ratio in T6‐OE‐WT cells demonstrated that TRMT6/61A‐mediated m 1 A modification of these tRNAs directly enhanced the translational efficiency of their cognate codons. (M) Schematic diagram of the ARG2 codon‐switch assay. To validate specificity, we engineered a synonymous mutant of ARG2 (ARG2‐MUT) by replacing the key cognate codons (GAC and GAG) with different, synonymous codons not recognized by the m 1 A‐modified tRNAs. aa, amino acid. (N) Western blot analysis comparing the protein expression of wild‐type ARG2 (ARG2‐WT) and the codon‐mutated version (ARG2‐MUT) in CTRL and OE‐WT cells, demonstrating that translation of ARG2‐WT is preferentially enhanced in OE‐WT cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Modification, Over Expression, Plasmid Preparation, Methylation, Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Staining, Knockdown, Luciferase, Construct, Activity Assay, Mutagenesis

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: Senescent tumor cells reprogram the tumor microenvironment in CRC (A) Uniform manifold approximation and projection (UMAP) visualization of integrated single‐cell transcriptomes from the CRC patients. The cell clusters with annotation of major cell types were shown (left top), and the feature plots showed the expression of TRMT6 , TRMT61A , ARG2, CDKN1A , and MTOR among cell types. (B) Chord diagrams illustrating the predicted intercellular communication network based on the number of interactions (top) and the overall interaction strength (bottom). Tumor cells were sub‐classified into ARG2 + and ARG2 ‐ subsets to reveal their distinct communication patterns. (C) Heatmaps provided an alternative visualization of the data in (B), quantifying the number of interactions (left) and interaction strength (right) between all identified cell populations. (D) Heatmaps visualizing the outgoing and incoming signaling patterns for key SASP‐related pathways, including GDF, HGF, CCL, TGFb, CXCL, IL1, and IGFBP. (e.g., HGF and IGFBP). The analysis identified ARG2 + cancer cells as prominent sources of these signals targeting M2 macrophages and fibroblasts. (E) In vitro validation of SASP gene expression. RT‐qPCR analysis revealed the relative mRNA fold change of a panel of key SASP factors in DLD1 (left) and HCT116 (right) cells. Expression in the TRMT6/61A‐overexpressing group (OE‐WT) is compared to the control group (CTRL). Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Single Cell, Expressing, In Vitro, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Control

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/61A‐induced STCs promote the malignant biological behaviors of recipient cancer cells. (A) Schematic diagram illustrating the experimental design. Conditioned medium (CM) was collected from cancer cells with stable overexpression of vector (CTRL) and wild‐type TRMT6/TRMT61A (OE‐WT), respectively. The two groups of CM were subsequently used to culture non‐transfected recipient CRC cells for functional assays. (B, C) The CM from OE‐WT cells enhanced the migration and invasion of recipient CRC cells. Representative images and quantification of transwell invasion (B) and wound‐healing (C) assays for DLD1 and HCT116 cells cultured with the two groups of CM are shown. Scale bars for (B), 100 µm. (D) CM from OE‐WT cells promoted the proliferation of recipient CRC cells. Real‐time cell proliferation curves for DLD1 and HCT116 cells were generated using the IncuCyte system. (E) Western blot analysis showing that CM from OE‐WT cells induced epithelial‐mesenchymal transition (EMT) in recipient DLD1 and HCT116 cells, as indicated by the upregulation of Snail, Slug, and N‐cadherin. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) TRMT6/61A‐overexpressing cancer cells promoted tumor growth in an in vivo model. The MC38 cells were co‐injected with either MC38‐derived CTRL or OE cells into BALB‐c/Nude mice ( n = 5 per group). The representative images of tumors and quantification of tumor weights were shown. (G) Western blot analysis of tumor lysates from the in vivo experiment (F) confirmed the expression of senescence and EMT markers. (H) Representative IHC images of tumor sections from the xenograft models, showing the abundance of m 1 A and expression of p16. Scale bars, 100 µm. (I) TRMT6/61A enhanced cancer stem cell (CSC)‐like properties. Representative bright‐field images and quantification of tumor spheres formed by DLD1 and HCT116 cells co‐cultured with CM derived from CTRL or OE‐WT cells. Scale bars, 50 µm. (J, K) Expression of stemness markers was upregulated in OE‐WT cells. RT‐qPCR analysis revealed increased NANOG and OCT4 mRNA in OE‐WT cells (J), and the Western blot analysis indicated upregulated EpCAM and CD44 protein levels in OE‐WT cells (K). Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Data are presented as mean ± SD from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Over Expression, Plasmid Preparation, Transfection, Functional Assay, Migration, Cell Culture, Generated, Western Blot, In Vivo, Injection, Derivative Assay, Expressing, Quantitative RT-PCR

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: TRMT6/61A‐induced STCs promote M2 macrophage polarization and cancer‐associated fibroblast (CAF) activation. (A) Conditioned medium (CM) derived from the TRMT6/61A‐overexpressing CRC cells (OE‐WT) promoted the migration of THP‐1 monocytes and primary colorectal CAFs compared with that derived from the vector group of CRC cells (CTRL). Representative images (left) and quantification (right) of a transwell migration assay are shown. (B) CM from OE‐WT cells induced the M2‐like polarization of THP‐1 monocytes. Representative flow cytometry plots (left) and quantification (right) showed an increased percentage of CD206‐positive cells after incubation with the indicated OE‐WT‐derived CM. (C, D) RT‐qPCR analysis of M2‐associated cytokine and chemokine expression in THP‐1 monocytes co‐cultured with CM derived from OE‐WT and CTRL groups of DLD1 (C) and HCT116 (D) cells. The expression levels were normalized by the expression in CTRL‐CM‐treated groups. (E) Western blot analysis showing the increased expression of CAF activation markers, including Fibroblast Activation Protein (FAP) and α‐Smooth Muscle Actin (α‐SMA), in CAFs cultured with CM derived from OE‐WT cells. Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots are representative of three independent biological replicates ( n = 3). (F) immunofluorescence assay confirmed the increased expression and co‐localization of FAP (green) and α‐SMA (red) in CAFs upon treatment with OE‐WT‐derived CM. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Activation Assay, Derivative Assay, Migration, Plasmid Preparation, Transwell Migration Assay, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Immunofluorescence

Journal: Advanced Science

Article Title: m 1 A‐Dependent TRMT6/61A‐ARG2 Axis Drives Protumorigenic Senescence by Remodeling the Tumor Microenvironment

doi: 10.1002/advs.202518536

Figure Lengend Snippet: NF‐κB/SASP is essential for pro‐tumorigenic effects of senescent CRC cells (A) Western blot analysis showing an activation of the NF‐κB pathway in TRMT6/61A‐overexpressing (OE‐WT) DLD1 and HCT116 cells compared with vector group (CTRL) cells, as indicated by increased levels of phosphorylated p65 (p‐p65). Relative densitometry quantification (normalized to GAPDH) is shown below the bands. Blots were representative of three independent biological replicates ( n = 3). (B) Schematic diagram of the experimental design for the NF‐κB inhibition experiments. CM was collected from OE‐WT cells pre‐treated with or without the NF‐κB inhibitor, BAY11‐7082. (C) RT‐qPCR analysis showing that NF‐κB inhibition abrogated the SASP gene signature in OE‐WT cells. (D, E, F) NF‐κB/SASP inhibition in STCs abolished its pro‐malignant effects on recipient cancer cells. Quantification and representative images were shown for the transwell invasion (D), real‐time cell proliferation (E), and wound‐healing (F) assays. The non‐transfected CRC cells were cultured with the CM derived from CTRL cells, OE‐WT cells, or OE‐WT cells pre‐treated with BAY11‐7082. Scale bars for (F), 100 µm. (G) NF‐κB/SASP inhibition rescued the STC‐induced M2 polarization. The flow cytometry showed that the CM from BAY11‐7082‐treated OE‐WT cells did not increase the proportion of CD206‐positive THP‐1 cells. (H,I) NF‐κB /SASP inhibition prevented STC‐induced activation of cancer‐associated fibroblasts (CAFs). Western blot analysis (H) and immunofluorescent assay (I) showed that CM from BAY11‐7082‐treated OE‐WT cells failed to upregulate the activation markers FAP (green) and α‐SMA (red). Relative densitometry quantification (normalized to GAPDH) is shown below the bands in (H). Nuclei were counterstained with DAPI (blue). Scale bars for (I), 20 µm. Data were presented as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human colorectal cancer cell lines DLD1 (RRID: CVCL_0248) and HCT116 (RRID: CVCL_0291) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) in October 2022.

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Inhibition, Quantitative RT-PCR, Transfection, Cell Culture, Derivative Assay, Flow Cytometry

Journal: Journal of Cancer

Article Title: Integrated Multi-omics Analyses Identify CDCA5 as a Novel Biomarker Associated with Alternative Splicing, Tumor Microenvironment, and Cell Proliferation in Colon Cancer Via Pan-cancer Analysis

doi: 10.7150/jca.91082

Figure Lengend Snippet: Correlation between CDCA5 and TME of colorectal cancer with TISCH database. (A) Correlation of CDCA5 with TME in CRC. (B, C) The annotation and distribution of the immune cell types in CRC GSE136394 and CRC GSE13955. The proportion of CDCA5 in different immune cell types in CRC GSE136394 dataset (D, E) and CRC GSE13955 dataset (F, G).

Article Snippet: The human colorectal cancer cell line DLD1 was purchased from Procell Life Science& Technology Co., Ltd (Wuhan,China), which was cultured in DMEM supplemented with 10% heat‐inactivated fetal bovine serum (FBS) (Sijiqing Biologic, Hangzhou, China) and was incubated at 37 ̊C in a humid incubator with air containing 5% CO2.

Techniques:

Journal: Journal of Cancer

Article Title: Integrated Multi-omics Analyses Identify CDCA5 as a Novel Biomarker Associated with Alternative Splicing, Tumor Microenvironment, and Cell Proliferation in Colon Cancer Via Pan-cancer Analysis

doi: 10.7150/jca.91082

Figure Lengend Snippet: Effect of CDCA5 silencing on DLD1 cell proliferation, apoptosis, migration and clone formation abilities. (A) mRNA level of CDCA5 after Si-CDCA5 transfection. (B) The viability of DLD1 cells after Si-CDCA5 transfection. (C) Cell clone formation after CDCA5 knockdown. (D) Cell proliferation after CDCA5 knockdown. (E) CDCA5-siRNA reduced the migration of colon cancer cells. (F) CDCA5-siRNA reduced the invasion of colon cancer cells. (G) Cell apoptosis after CDCA5 knockdown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

Article Snippet: The human colorectal cancer cell line DLD1 was purchased from Procell Life Science& Technology Co., Ltd (Wuhan,China), which was cultured in DMEM supplemented with 10% heat‐inactivated fetal bovine serum (FBS) (Sijiqing Biologic, Hangzhou, China) and was incubated at 37 ̊C in a humid incubator with air containing 5% CO2.

Techniques: Migration, Transfection, Knockdown