Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: A hypoxia-characteristic cluster identified by Microwell-seq exhibited pronounced ferroptosis resistance in MSS CRC cells. (A and B) t-distributed stochastic neighbor embedding (t-SNE) plot of Microwell-seq analysis based on gene expressions of SW480 and WiDr. (C and D) Comparative gene set enrichment analysis (GSEA) of signaling pathways in different clusters. The red color represents up-regulation and blue represents down-regulation, calculated with the formula: ± Log2|NES/p.adjust|. Grey color represents no enrichment in the indicated pathway. NES: normalized enrichment score. (E and F) GSEA analysis showed the indicated pathway activity between the hypoxia cluster and other clusters. (G and H) Correlation analysis of hypoxia scores, glycolysis scores, and ferroptosis suppressor scores in WiDr and SW480 cells was performed using Pearson's method. (I and J) The ferroptosis suppressor score of SW480 and WiDr with DMSO or RSL3 treatment was analyzed by AddModuleScore tool. P values were calculated by Wilcox.test. (K and L) The ferroptosis suppressor score of hypoxia cluster in SW480 and WiDr treated with DMSO or RSL3 was shown. P values were calculated by Wilcox.test.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Protein-Protein interactions, Activity Assay

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: HIF-1α nuclear distribution increased in RSL3-resistant CT26 cells and significantly promoted tumourigenicity and metastasis. (A) The diagram demonstrated the procedure for sphere formation. (B and C) The representative images of sphere formation in MSS CRC cells and quantification analysis of sphere number derived from SW480, HT-29, and WiDr. (D) HIF-1α expression in WiDr was detected by western blotting. (E) qPCR analyzed the indicated gene expression involved in glycolysis in WiDr spheres. (F) Cell viability of parental CT26 and RSL3-resistant CT26 (Re-CT26). (G) Cytoplasm and nuclear HIF-1α expression in CT26. α-tubulin was used as an internal reference for the cytoplasm, and Histone 3 was used as a nuclear reference. (H) qPCR analyzed the indicated gene expression involved in glycolysis in CT26. (I) Image of subcutaneous tumors derived from parental CT26 and Re-CT26. (J) Tumor volume determined by formula: 0.52 × Long × width 2 . (K)Tumor weight of subcutaneous tumors. (L) Bioluminescent images in liver metastatic models. (M) Images of liver metastasis derived from parental CT26 and Re-CT26. (N) Number of liver nodules in liver metastasis models. (O) Representative hematoxylin and eosin (H&E) and immunohistochemical staining images from liver metastasis models. Scale bar: 25 μm. (P) Quantification of immunohistochemical staining results shown in (O). Data are shown as means ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns: not significant. Two-way ANOVA in (J), others unpaired two-tailed Student's t -test.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Western Blot, Gene Expression, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: P4HA1 was a major factor regulated by HIF-1α and was enriched in ferroptosis-resistant cells. (A) Venn diagram screening 10 genes commonly induced by hypoxia and the glycolysis pathway in SW480 and WiDr by single-cell sequencing. (B) Gene expression heatmap showed the distribution of genes in different clusters in the presence or absence of RSL3. D: DMSO, R: RSL3. (C) Correlation between ferroptosis suppressor score and P4HA1 in MSS CRC containing 119 patients using Pearson's method. (D) Kaplan-Meier plots of RFS in MSS colon cancer patients according to P4HA1 expression. (E) The correlation between HIF-1α and P4HA1 was evaluated by Spearman's analysis in a colon adenocarcinoma cohort of 457 patients. (F) Relative P4HA1 mRNA expression after HIF-1α knockdown, detected by qPCR. (G) Relative P4HA1 protein expression after HIF-1α knockdown, detected by Western blot. (H) Relative P4HA1 mRNA expression after HIF-1α overexpression, detected by qPCR. (I) Relative P4HA1 protein expression after HIF-1α overexpression, detected by Western blot. (J) Relative P4HA1 expression, detected by qPCR. (K) HIF-1α binding motif predicted from JASPAR. (L) The prospective binding site of HIF-1α on the promoter of P4HA1. (M) ChIP assay of HIF-1α and IgG in parental CT26 cells or Re-CT26 cells, followed by qPCR for the binding sequences.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Single Cell, Sequencing, Gene Expression, Expressing, Knockdown, Western Blot, Over Expression, Binding Assay

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: HIF-1α inhibition enhanced ferroptosis inducer sensitivity in MSS CRC cells. (A and B) Cell viability of HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment. (C and D) DCFH-DA oxidation in HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment were quantified using flow cytometry with DCFH-DA probe. (E and F) Lipid peroxidation levels of HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment were detected using flow cytometry with C11-BODIPY 581/591 (FITC channel; excitation/emission: 488/510 nm). (G) MDA levels in cells subjected to the indicated treatment. (H) GSH levels in cells subjected to the indicated treatment. Results are shown as means ± SD. ∗ P < 0.05 ; ∗∗ P < 0.01 ; ∗∗∗ P < 0.001 ; ∗∗∗∗ P < 0.0001 . P values were calculated by one-way ANOVA.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Inhibition, Flow Cytometry

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: In vitro cytotoxicity of CIK cells against HCT-15 CRC cells at various E:T ratios. The percentage of viable tumor cells was normalized to their respective control. Data are presented as mean ± SD from technical triplicates. Statistical analysis was assessed using one-way ANOVA. **** p < 0.0001.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: In Vitro, Control

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: Cytotoxic effects of 5-FU and CIK cells on HCT-15 cells. HCT-15 were pre-treated with 5-FU (0.050, 0.075, and 0.100 mg/mL) for 24 hours, followed by co-culture with CIK cells for 72 hours at E:T ratios of 1:5, 1:1, 10:1, and 20:1. ( A ) Cytotoxic effect of 5-FU on HCT-15 cells. ( B ) Cytotoxic effect of CIK cells at various E:T ratios on HCT-15 cells. ( C ) Combined effects of 5-FU and CIK cells on HCT-15 cells. Statistical analyses for ( A and B ) were performed using one-way ANOVA, while ( C ) was analyzed using two-way ANOVA. Data are presented as mean ± SD from technical triplicates. * p < 0.05, *** p < 0.001, **** p < 0.0001, compared with the control group.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Co-Culture Assay, Control

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: In vitro cytotoxic activity of CIK cells against HCT-15 cells. The cytotoxic effect of CIK cells expanded from healthy donor 3 versus CRC patient was assessed at E:T ratios of 1:5, 1:2, 1:1, 5:1, 10:1, 20:1 and 40:1. Cancer cells without CIK cells served as normalizing controls for both healthy donor and cancer patient. Statistical analysis was assessed using independent t -test. Data are presented as mean ± SD from technical triplicates. ** p < 0.01, **** p < 0.0001, compared to the respective control.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: In Vitro, Activity Assay, Control

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: The relative levels of cytokines in the conditioned media of ( A ) CIK cells from donor 3 and ( B ) HCT-15 cells were visualized using a human cytokine antibody array (Proteome Profiler Human XL Cytokine Array Kit, R&D Systems). The following highly expressed markers in CIK cells and HCT-15 cells culture are indicated by the boxes on the membranes: A: Adiponectin; B: Apolipoprotein A-I; C: Angiogenin; D: Complement component C5/C5a; E: Fas ligand (FasL); F: Granulocyte macrophage colony-stimulating factor (GM-CSF); G: Interleukin-2 (IL-2); H: Interleukin-5 (IL-5); I: Interleukin-8 (IL-8); J: Macrophage inflammatory protein-1 alpha/Macrophage inflammatory protein-1 beta (MIP-1α/MIP-1β); K: Platelet factor 4 (PF4); L: Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES); M: Retinol binding protein 4 (RBP-4); N: Cluster of differentiation 31 (CD31); O: T-cell immunoglobulin and mucin domain 3 (TIM-3); P: Dickkopf-related protein 1 (DKK-1); Q: Growth differentiation factor 15 (GDF-15); R: Human interferon-inducible protein 10 (IP-10); S: Lipocalin-2; T: Monokine induced by gamma interferon (MIG); U: Serpin E1; V: Vascular endothelial growth factor (VEGF). Differences in spot intensity reflect relative cytokine expression levels, where darker spots indicate higher expression and lighter spots indicate lower expression. Background signal was subtracted during image analysis.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Ab Array, Activation Assay, Binding Assay, Expressing

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: Differentially expressed cytokines and growth factors in the conditioned medium of CIK cells from donor 3 alone, HCT-15 cells, and co-culture of CIK cells and HCT-15 cells at E:T ratios of 1:1 and 10:1. ( A ) Heatmap of 105 cytokines and chemokines. ( B ) Venn diagram depicting markers that were highly expressed (pixel density >300,000) in CIK cells and HCT-15 cells.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Co-Culture Assay

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: Distinct protein-protein interaction among the significantly regulated cytokines and growth factors within ( A and B ) CIK cells from donor 3 or ( C and D ) HCT-15 cells during co-culture, as elucidated by STRING. ( A ) Functional and physical associations of cytokines significantly regulated within CIK cells. ( B ) Association strengths of these regulated factors in different biological processes. Red: positive regulation of mononuclear cell proliferation; Green: positive regulation of immune system process; Blue: leukocyte migration, Yellow: negative regulation of cellular process; Pink: positive regulation of cellular process. ( C ) Functional and physical associations of cytokines significantly regulated within HCT-15 cells. ( D ) Association strengths of these regulated factors in different biological processes. Yellow: positive regulation of immune system process; Green: positive regulation of the MAPK cascade; Red: regulation of cell population proliferation; Pink: regulation of cell communication. The networks were generated using the STRING database. Nodes represent proteins, and edges indicate predicted or known associations. These interactions reflect database-derived relationships and suggest potential functional connectivity but do not imply direct causal interactions.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Co-Culture Assay, Functional Assay, Migration, Generated, Derivative Assay

Journal: ImmunoTargets and Therapy

Article Title: An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

doi: 10.2147/ITT.S594441

Figure Lengend Snippet: Levels of cytokines and growth factors in HCT-15 cells alone and co-cultured with CIK cells from donor 3 at E:T ratios of 1:1 and 10:1. The factors are grouped based on their functions into ( A ) antitumor, ( B ) immunosuppression, and ( C ) cell proliferation and metastasis. The pixel intensity is shown as mean ± SD from technical triplicates. Two-way ANOVA followed by post-hoc Tukey analysis was performed. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared to HCT-15 cells; ** p < 0.01, **** p < 0.0001 compared to CIK:HCT-15 at an E:T ratio of 1:1.

Article Snippet: The human colorectal cancer (CRC) cell line HCT-15 was obtained from the American Type Culture Collection (ATCC; USA).

Techniques: Cell Culture