Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 1. LC cells secrete lactate to promote the activation of CAFs. A, a diagram for the co-culture system of green fluorescent protein (GFP)-labeled mLFs (COL2A1+ACTA2+) with TC-1 cells; B, mRNA and protein levels of active CAF markers (TGFB1 and SDF1) in the mLFs after co-culture with TC-1 cells determined by RT-qPCR or WB analysis; C, a diagram for the co-culture system of NHLFs with lung cancer cell lines (H1299 and H460); D, mRNA expression of active CAF markers (TGFB1 and SDF1) in the NHLFs after co-culture examined by RT-qPCR; E-F, mRNA (E) and protein (F) levels of TGFB1 and SDF1 in NHLFs cultured in LE-CM (CM of LE cells [BEAS-2B or HBE]) determined by RT-qPCR or WB analysis; G-H, mRNA (G) and protein (H) levels of TGFB1 and SDF1 in NHLCs cultured in the <3 kDa and >3 kDa fractions of the CM of H1299 cells, determined by RT-qPCR or WB analysis; I-J, mRNA (I) and protein (J) levels of TGFB1 and SDF1 in NHLCs cultured in the CM of H1299 cells upon boiling for 5 min or 15 min, determined by RT-qPCR or WB analysis; K, lactate secretion by LE cell lines (BEAS-2B and HBE) and by LC cell lines (H1299, H460, A549, and CALU-3); L, a diagram for LE-CM or LC-CM with the addition of MCT inhibitors (AR-C155858 or 7ACC2) for NHLF culture; M, mRNA and protein levels of TGFB1 and SDF1 in NHLCs cultured in LC-CM alongside MCT inhibitors determined by RT-qPCR and WB analysis. Differences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed. ***p < 0.001, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Activation Assay, Co-Culture Assay, Labeling, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 3. NUSAP1 promotes nuclear translocation of NUSAP1 to activate the JUNB-FRA1-FRA2-DESMIN axis. A, three-cluster (C1, C2, C3) heatmap of ATAC-seq peaks, ±2 kb from transcription start site in NHLF cells treated with lactate for 48 h or not; B, HOMER analysis of transcription factor (TF)-enriched promoter regions (1–2 kb) in NHLF cells treated with lactate; C, genome browser view of DESMIN promoter, chromatin accessibility (ATAC-seq), and RNA-seq profiles in NHLF cells treated with lactate for 48 h or not; D, JUNB-driven luciferase activity in NHLFs treated with lactate for 48 h or not; E, DESMIN promoter-driven luciferase in NHLFs, transduced with the indicated plasmids, and treated with lactate (24 mM) for 48 h; F-G, mRNA (F) and protein (G) levels of DESMIN in NHLF cells after knockout of different JUNB segments, in the presence of lactate treatment or not, determined by RT-qPCR and WB analysis; H, luciferase assay to examine the binding between JUNB and FRA1, FRA2, c-FOS, and c-JUN; I-J, mRNA (I) and protein (J) levels of DESMIN in NHLFs after FRA1 or FRA2 knockdown, in the presence of lactate treatment or not, determined by RT-qPCR and WB analysis. Differences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed. ***p < 0.001, ****p < 0.0001.

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Translocation Assay, RNA Sequencing, Luciferase, Activity Assay, Transduction, Knock-Out, Quantitative RT-PCR, Binding Assay, Knockdown

Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 4. Lactate promotes nuclear translocation of NUSAP1 to activate the JUNB-FRA1-FRA2-DESMIN axis. A, diagram presentation of the process of lactate-mediated nuclear translocation of JUNB-FRA1-FRA2 transcriptional complex and the regulation on DESMIN transcription determined by RT-qPCR and WB analysis; B–C, mRNA and protein levels of DESMIN in NFLFs after co-culture with lactate (24 mM) for different duration; D-E, mRNA (D) and protein (E) expression of NUSAP1 and DESMIN in NHLFs determined by RT-qPCR and WB analysis; F, luciferase activity of DESMIN in NHLFs under different conditions determined by promoter luciferase reporter gene assay; G-I, ChIP-PCR analysis of DESMIN promoter (JUNB-B) occupancy by FRA1 or FRA2 in NHLF cells transduced with the indicated siRNAs. J ~ K, Immunofluorescence for gamma-H2AX, and EdU assay for DNA replication in NHLF cells transduced with the indicated siRNAs. Differences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed. ***p < 0.001, ****p < 0.0001.

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Translocation Assay, Quantitative RT-PCR, Co-Culture Assay, Expressing, Luciferase, Activity Assay, Reporter Gene Assay, Transduction, Immunofluorescence, EdU Assay

Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 5. JUNB control of the DESMIN promoter in CAFs is critical for LC tumor growth and invasion. A–C, Experimental design, migration, and invasion of H1299 cells co- cultured with sgC or sgJUNB-B NHLF for 16 h. D–E, Experimental design and H&E staining of organotypic gels combining H1299 cells with sgC or sgJUNB-B NHLF. F–G, Subcutaneous xenograft co-implantation in NSG mice (male, 7 weeks old) of H1299 cells with sgC or sgJUNB-B NHLF cells. H–I, Immunohistochemistry (IHC) for Masson’s trichrome, HA, and αSMA in xenografts. Differences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed in cell experiments. In F ~ I, each group contains 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Control, Migration, Cell Culture, Staining, Immunohistochemistry

Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 6. NUSAP1 upregulation mimics lactate loss of DESMIN upregulation and promotes CAF activation. A–C, Experimental design and indicated genes in NHLF cells transduced with the indicated overexpression vectors. D–H: Experimental design, proliferation, DNA replication, migration, and invasion of H1299 cells co-cultured with NHLF cells transduced with the indicated overexpression vectors. I–J, Subcutaneous xenograft co-implantation of H1299 cells with NHLF cells transduced with the indicated overexpression vector in NSG mice (male, 7 weeks old). K, Immunohistochemistry (IHC) for Masson’s trichrome, HA, and αSMA in xenografts. Dif ferences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed in cell experiments. In I ~ K, each group contains 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Activation Assay, Transduction, Over Expression, Migration, Cell Culture, Plasmid Preparation, Immunohistochemistry

Journal: Redox biology

Article Title: Lactate-induced activation of tumor-associated fibroblasts and IL-8-mediated macrophage recruitment promote lung cancer progression.

doi: 10.1016/j.redox.2024.103209

Figure Lengend Snippet: Fig. 7. IL-8 Secretion by DESMIN + CAFs Promotes Activation and Infiltration of TAMs. A–B, Experimental design and ELISA for NHLF secretion cytokines incubated with lactate in a dose-dependent manner. C, Subcutaneous xenograft co-implantation in B6 mice (male, 7 weeks old) of 3LL cells with mLF cells. D, the TAMs counts in 3LL and mLF derived xenografts by FACS. E, mRNA levels of indicated genes in mouse tumor tissues. F–G, mRNA levels or protein content of Th1 or Th2 related genes in mouse tumor tissues. H–I, mRNA level and immunohistochemistry of TAMs macrophages in mouse tumor tissues. J ~ K, number of infiltrated TAMs, and the expression of neoplastic PD-L1 in the mouse tumor tissues on the 6th, 9th and 12th months was determined by flow cytometry. L–N, Experimental design and ELISA and qPCR assays for mRNA and protein content of indicated genes in PMA-incubated THP-1 cells. ˜O P, Expriment design and migration of PMA-treated THP-1 cells towards the supernatant of NHLF cells examined by the Transwell assay with or without IL-8R antagonism. Differences were analyzed by Student’s t-test or ANOVA, after ANOVA, Tukey’s post-hoc multiple analysis were used. Three biological replicates were performed in cell experiments. In C ~ K, each group contains 6 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For MCT inhibition, NHLF cells were treated with either 10 μM of AR-C155858 or 7ACC2 (Selleck Chemicals) for 48 h. To inhibit protein synthesis, NHLF cells were treated with 50 μg/mL of cycloheximide (Sigma).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay, Immunohistochemistry, Expressing, Flow Cytometry, Migration, Transwell Assay

Journal: Cancers

Article Title: Conventional Cancer Therapies Can Accelerate Malignant Potential of Cancer Cells by Activating Cancer-Associated Fibroblasts in Esophageal Cancer Models

doi: 10.3390/cancers15112971

Figure Lengend Snippet: Efficacy of chemotherapy or radiotherapy against cancer cells and fibroblasts. ( A , B ) Viability of human-derived fibroblasts ( A ) or cancer cells ( B ) induced by chemotherapy (CDDP or 5-FU) was measured using XTT metabolic activity. The control group (100 %) did not receive chemotherapy ( n = 4. Mean ± SD). ( C ) Viability of TE4 and FEF3 cells induced by radiotherapy measured using XTT ( n = 4. Mean ± SD). ( D ) Relative proliferation curves of FEF3 cells treated by radiotherapy. Day 0 is set as the control ( n = 4; mean ± SD). In ( A – C ), the representative examples from five experiments were shown, and the representative example from three experiments was shown in ( D ).

Article Snippet: NHLF, a normal human lung fibroblast cell line, was purchased from Cambrex Corporation (East Rutherford, NJ, USA), and WI-38, a human fetal lung fibroblast cell line, was purchased from the Health Science Research Resource Bank (Osaka, Japan).

Techniques: Derivative Assay, Activity Assay

Journal: Cancers

Article Title: Conventional Cancer Therapies Can Accelerate Malignant Potential of Cancer Cells by Activating Cancer-Associated Fibroblasts in Esophageal Cancer Models

doi: 10.3390/cancers15112971

Figure Lengend Snippet: Phenotypic changes in normal fibroblasts after chemotherapy. ( A ) Treatment schedule. ( B ) Fluorescent microscopic images of human fibroblasts (FEF3, NHLF, and WI-38) after chemotherapy (CDDP or 5-FU). FAP (green), α-SMA (red), and DAPI (blue) staining is shown. Scale bar, 20 µm. The representative examples from four experiments were shown. ( C , D ) Flow cytometric analysis of FAP and α-SMA expression in the three fibroblast cell lines. Representative flow cytometric histograms are shown ( C ), and the relative fluorescence intensity (RFI) was calculated (( D ), n = 4. Mean ± SD).

Article Snippet: NHLF, a normal human lung fibroblast cell line, was purchased from Cambrex Corporation (East Rutherford, NJ, USA), and WI-38, a human fetal lung fibroblast cell line, was purchased from the Health Science Research Resource Bank (Osaka, Japan).

Techniques: Staining, Expressing, Fluorescence

Journal: Cancers

Article Title: Conventional Cancer Therapies Can Accelerate Malignant Potential of Cancer Cells by Activating Cancer-Associated Fibroblasts in Esophageal Cancer Models

doi: 10.3390/cancers15112971

Figure Lengend Snippet: Phenotypic changes in fibroblasts following radiotherapy. ( A ) Treatment schedule. ( B ) Fluorescent microscopic images of human-derived fibroblasts (FEF3, NHLF, and WI-38) after radiotherapy (16 Gy/8 fx). FAP (green), α-SMA (red), and DAPI (blue) staining is shown. The Sham group is used as the control group. Scale bar, 20 µm. The representative examples from four experiments were shown. ( C , D ) Flow cytometric analysis of FAP and α-SMA expression in the three fibroblast cell lines. Representative flow cytometric histograms are shown ( C ), and the RFI was calculated (( D ), n = 4. Mean ± SD).

Article Snippet: NHLF, a normal human lung fibroblast cell line, was purchased from Cambrex Corporation (East Rutherford, NJ, USA), and WI-38, a human fetal lung fibroblast cell line, was purchased from the Health Science Research Resource Bank (Osaka, Japan).

Techniques: Derivative Assay, Staining, Expressing