Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 7 The number of T cells migrating across the transwell and the protein expression levels of chemokines mediating T-cell recruitment. (A) The activat ed CD3+ T cells were seeded into transwells with simple media and conditioned media from T24 cell cultures, NK cell cultures, and NK and T24 cell cocul tures. Summary data of the cumulative numbers of CD3+ T cells that migrated across the transwell in each condition are shown (n = 3 per group). ns, not significant; *p < 0.05 ; (B) Protein expression levels of chemokines in the supernatant from T24 culture, NK culture, and NK + T24 coculture measured using ELISA (n = 3 per group). Data expressed as mean ± SD were plotted. Abbreviations: CCL: C-C-motif ligand; CXCL:C-X-C-motif ligand; XCL1:X-C-motif ligand

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 8 Receptors of the chemokines expressed on CD3+ T cells and T cell count migrating across the transwell after blocking CCL1/2/20 in the superna tant of coculture medium. (A) The expressions of the chemokine receptors on CD3+CD56− T cells were detected using flow cytometry (n = 3 for all mol ecules). The representative images are shown. (B) Summary data of the cumulative numbers of T cells determined using a fluorescence cell analyzer after being harvested from the lower chambers, which were added into blocking antibodies against CCL1 (0.25 µg/mL), CCL2 (40 ng/mL), or CCL20 (10 µg/ mL) (n = 3 for each). Data are shown as mean ± SD. Statistical significance was determined using an unpaired t-test. *p < 0.05. Abbreviations: CXCR: C-X-C chemokine receptor; CCR:C-C chemokine receptor; XCR:X-C chemokine receptor

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Cell Counting, Blocking Assay, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A, B: the expression of HOTAIR and CCL22 mRNA were detected by RT-qPCR. C: CCL22 protein analyzed by ELISA assay. ****, P<0.001.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A: The HOTAIR and CCL22 mRNA expression examined by RT-qPCR B: Cell apoptosis detected by Flow cytometry. C: The cell proliferation tested by MTS assay. D: The CCL22 protein expression tested by ELISA. E: The cell migration and invasion detected by Transwell assay. **, P<0.05; ***, P<0.01; ****, P<0.001;ns, no significance.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, MTS Assay, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer-associated fibroblasts promote pro-tumor functions of neutrophils in pancreatic cancer via IL-8: potential suppression by pirfenidone

doi: 10.1007/s00262-025-03946-z

Figure Lengend Snippet: IL-8 secreted by activated neutrophils is suppressed by PFD. A IL-8 secretion by neutrophils measured by ELISA. IL-8 secretion was significantly increased in neutrophils stimulated with CAF-CM compared to non-stimulated neutrophils. PFD administration during neutrophil activation with CAF-CM significantly suppressed IL-8 secretion. B Effect of secretions from activated neutrophils on cancer cell proliferation. The presence of neutrophils significantly increased MIA PaCa-2 proliferation. Mean ± SD is shown. Each dot represents the number of cells per well. Student’s t-test was used. ***, P < 0.001. C Pancreatic cancer cells induce fibroblasts to become iCAFs, and iCAF-CM activates neutrophils, increasing their IL-8 secretion and enhancing the malignant traits of pancreatic cancer cells. Conversely, PFD acts on activated neutrophils, suppressing the malignant traits of pancreatic cancer cells. This figure was created with BioRender.com

Article Snippet: To evaluate IL-8 levels in neutrophil secretions, culture supernatants were assayed using a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, KE00006) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: CXCL14 silencing is associated with adverse prognosis of CRC. ( A ) CRC datasets show CXCL14 downregulation in cancer development and metastasis (mean + SEM). * p < 0.05, *** p < 0.001. ( B ) The ProteinAtlas database demonstrates that CXCL14 is highly expressed in normal colon (( i ), PID3753) and rectal (( iv ), PID:3243) gland cells (arrow), but significantly reduced in colonic adenocarcinoma tissue (( ii ), PID:2002; ( iii ), PID:1958) and rectal adenocarcinoma (( v ), PID:1424; ( vi ), PID:1416). The bar represents 50 μm. ( C ) CXCL14 is silenced in human colon cancer cell lines (data from GTEx database) compared to normal tissue (data from ProteinAtlas database). ( D ) Mouse colon tissue and cell line qPCR results show very low CXCL14 in MC38 and CT26 cells. ( E ) Clinical datasets reveal CXCL14 negatively correlates with EMT markers in colon cancer ( p < 0.01). ( F ) CXCL14 exhibits a general negative correlation with the tumor cell proliferation marker TK1 in the four CRC datasets. ( G ) Kaplan–Meier curve of post-progression survival (PPS) for colon cancer patients stratified by CXCL14 expression (probe 222484_s_at). ( H ) CXCL14 strongly correlates with T cell (0.4 < r < 0.7) and NK cell markers (r > 0.6) in GTEx colon tissue datasets, but weakly or not at all with Treg cells, neutrophils, and macrophages (r < 0.2). ( I ) Analysis of the correlation between CXCL14 expression levels and the expression levels of MMP family genes in the Sidra-LUMC dataset.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Marker, Expressing

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Overexpression of CXCL14 inhibits proliferation of colon cancer cells. ( A ) RNA-seq data ( n = 2), q-PCR ( n = 3) and WB result of CXCL14 expression in the non-induced and DOX-induced MC38-TetOn-CXCL14 cell line. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, t -test. ( B ) Proliferation curves of control and 0.5 and 1 μg/mL DOX-induced MC38 cell ( n = 6). Data represent the mean ± SD. *** p < 0.001, one-way ANOVA. ( C ) RNA-seq data of TK1 mRNA of control and CXCL14 -overexpressed MC38 cell ( n = 2). Data represent the mean ± SD. * p < 0.05, t -test. ( D ) WB detection, qPCR data of CXCL14 expression (left) and proliferation curves of parental and CXCL14-overexpressed CT26 cells. Data represent the mean ± SD. *** p < 0.001, t -test and one-way ANOVA. ( E ) WB assay and qPCR data of CXCL14 expression (left) and proliferation curves of parental and CXCL14 -overexpressed HCT15 cells. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001, t -test and one-way ANOVA. ( F ) The proliferation inhibition effect of recombinant human CXCL14 (rhCXCL14) on colon cancer cell line in vitro. **** p < 0.0001, one-way ANOVA. ( G ) The tumor growth curves of DOX-induced and non-induced groups of C57bl/6n mice with subcutaneous MC38-plvx-TetOn-CXCL14 cells. The tumor volume of the DOX-induced group was significantly lower than that of the non-induced group ( n = 8), * p < 0.05, ** p < 0.01, t -test. ( H ) Appearance of dissected subcutaneous tumor tissue ( n = 8 for control and DOX+ group). ( I ) The tumor tissue was excised from the subcutaneous tissue and weighed. The tumor weight of the DOX-induced group was significantly lower than that of the non-induced group ( n = 8), ** p < 0.01, t -test. ( J ) The body weight curves of non-induced and DOX-induced mice ( n = 8), no significant differences by t -test.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Over Expression, RNA Sequencing, Expressing, Control, Inhibition, Recombinant, In Vitro

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: RNA-seq analysis of the regulatory role of CXCL14 overexpression on the transcriptome of MC38 cells. ( A ) The volcanic plot displayed that the induction of CXCL14 expression led to a significant upregulation of 625 genes and downregulation of 1237 genes ( p -adj < 0.05) in MC38 cells, representing approximately 15% of the total genes. ( B ) The Gene Ontology (GO) analysis of differentially expressed genes (DEGs) between the non-induced and DOX-induced MC38-TetOn-CXCL14 cell line. ( C ) GSEA showed that CXCL14 significantly suppressed cell EMT signaling. Dot size indicates the number of genes counted in each term. ( D ) RNA-seq data of EMT-related genes expression of MC38-TetOn-CXCL14 cell line prior or post DOX induction (all p -values < 0.01). ( E ) The GSEA analysis of the impacts of CXCL14 overexpression on MC38 cell migration, locomotion and chemotaxis. ( F ) RNA-seq data of MMP family genes of non-induced and DOX-induced MC38-TetOn-CXCL14 cell line ( n = 2). Here only genes with non-zero values are displayed. family. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: RNA Sequencing, Over Expression, Expressing, Migration, Chemotaxis Assay

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Overexpression of CXCL14 inhibits invasion of colon cancer cells. ( A ) The invasion assay of non-induced and DOX-induced CXCL14-overexpressed MC38 cells ( n = 3). Data represent the mean ± SD. * p = 0.011, t -test. ( B ) The invasion assay demonstrates that overexpression of CXCL14 in CT26 cells impairs their invasion capability ( n = 3), * p < 0.05, t -test. ( C ) The invasion assay has revealed that the restored expression of CXCL14 in HCT15 cells effectively suppresses their invasion capacity. * p <0.01, t -test. ( D ) The wound repair assay of control and 1 μg/mL DOX-induced MC38 cell ( n = 10). The red lines indicate the leading edge of the migrating cells. Data represent the mean ± SD. *** p < 0.001, t -test. ( E ) The wound repair assay of parental and CXCL14-overexpressed-HCT15 cell lines ( n = 6). Data represent the mean ± SD. ** p < 0.01, one-way ANOVA. ( F ) WB assay of the effect of CXCL14-conditioned medium on the phosphorylation of Erk, Akt, and p38 proteins in MC38 cells. ( G ) The grayscale quantification results of the WB bands in ( F ). the data represents the consistent outcomes from multiple replicates of the experiment. ( H ) qPCR data of EMT-related genes in parental and CXCL14-overexpressed CT26 cells ( n = 2), * p < 0.05, ** p <0.01, t -test. ( I ) The appearance of the liver 21 days after spleen inoculation with MC38. ( J ) The liver weight data, n = 5, * p = 0.02. ( K ) Number of metastatic tumor foci on the liver surface. ** p < 0.01, n = 5.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Over Expression, Invasion Assay, Expressing, Control, Phospho-proteomics

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Transcriptome analysis showed that CXCL14 regulates the tumor microenvironment. ( A ) Frozen sections from mice bearing subcutaneous tumors revealed the presence of the reporter gene EGFP, indicating the induction of CXCL14 expression within the tumor. ( B ) RNA-seq data of the expression level of CXCL14 in non-induced and DOX-induced CXCL14 overexpression tumor tissues ( n = 4), * p < 0.05, t -test. ( C ) RNA-seq results revealed that there were 720 significantly upregulated genes and 234 downregulated genes in CXCL14 OE MC38 subcutaneous tumors compared to MC38 non-induced tumors. ( D ) The GO analysis results indicate that the gene changes caused by CXCL14 overexpression are mainly concentrated in immune/inflammatory response, chemotaxis, and T cell-related signal transduction. ( E ) The KEGG enrichment analysis results indicate that the gene changes caused by CXCL14 overexpression are mainly concentrated in immune deficiency, Th cell differentiation, and antigen presentation. ( F ) The RNAseq data of T cell marker genes in the subcutaneous tumor with non-induced and DOX-induced CXCL14 overexpression ( n = 4), * p < 0.05, t -test. ( G ) The heat map of qPCR data of dendritic cell markers in the subcutaneous tumor with non-induced and DOX-induced CXCL14 overexpression ( n = 4), p < 0.05, t -test. ( H ) GSEA of hallmark inflammatory and IFNγ gene signatures was performed to compare CXCL14-overexpressed MC38 cells with non-induced MC38 cells.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Expressing, RNA Sequencing, Over Expression, Chemotaxis Assay, Transduction, Cell Differentiation, Immunopeptidomics, RNA sequencing, Marker

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: CXCL14 enhances tumor T cell infiltration. ( A ) The H-E staining of the tumor showed that there were more necrotic areas in the tumor overexpressing CXCL14. ( B ) The abundance of CD3 cells was significantly increased in the tumor tissues induced by DOX+ overexpression of CXCL14. Each treatment had 4 biological replicates, and 5 high-power fields were randomly selected from each tumor to count the positive cells. ( C ) The CD3 (PE-Cy5.5) and SSC scatter plot of the tumor cell suspension. The gate in the figure selects CD3-positive T lymphocytes. ( D ) The flow cytometry results of CD4 (FITC) vs. CD8 (APC). The Q1 quadrant represents CD4+CD8-T lymphocytes, and the Q3 quadrant represents CD4-CD8+ T lymphocytes. ( E ) The statistical results of the Q1 quadrant cells in ( D ) show that CD4+CD8- T lymphocytes were significantly increased in the tumor cells induced by DOX overexpression ( p < 0.001). ( F ) The statistical results of the Q3 quadrant cells in ( D ) showed that CD4-CD8+ T lymphocytes were significantly increased in the tumor cells induced by DOX overexpression ( n = 4), * p < 0.05, *** p < 0.001, t -test. ( G ) The scatter plot of FSC/SSC of the tumor cell suspension. The gate encloses live cells. ( H ) The scatter plot of CD40/APC and CD11b/PC7 of tumor cells. The Q2 quadrant represents double-positive cells, which are tumor macrophages. Colors indicate the relative cell density in each region of the plot. ( I ) There was no significant change in the content of macrophages in tumor-infiltrating cells ( n = 4), t -test.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Staining, Over Expression, Suspension, Flow Cytometry