human mss crc cell lines sw480  (ATCC)

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: Journal for Immunotherapy of Cancer

Article Title: Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target

doi: 10.1136/jitc-2021-004302

Figure Lengend Snippet: Identification of two distinct tumor microenvironment-based immune classes in pMMR/MSS CRC. An immune class and a non-immune class were identified using consensus clustering in the (A) GSE39582 cohort and (C) COAD cohort. In the heatmap, high and low ssGSEA scores of 13 immune cell or immune response signatures are represented in yellow and blue, respectively. Box plots showing expression of ICB response-related signatures between immune class, non-immune class and dMMR/MSI samples in the (B) GSE39582 cohort and (D) COAD cohort. **P<0.01, ****p<0.0001 vs control group. COAD, colon adenocarcinoma; CRC, colorectal cancer; dMMR/MSI, deficient-mismatch-repair or microsatellite instability; GEP, gene expression profile; ICB, immune checkpoint blockade; pMMR/MSS, proficient-mismatch-repair/microsatellite stability; ssGSEA, single-sample gene-set enrichment analysis.

Article Snippet: Two human MSS CRC cell lines (HT29 and SW480) and a murine MSS CRC cell line (CT26) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Control, Gene Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target

doi: 10.1136/jitc-2021-004302

Figure Lengend Snippet: Identification of GBP2 as a gene of interest. (A) Venn diagram of intersection of differentially expressed genes (DEGs) and RNA binding proteins (RBPs) in the MSS samples of COAD cohort. The intersected genes were further screened by Univariate Cox analysis. The cut-off criteria of DEGs were log2 fold change >0.585 and an adjusted p<0.05. (B) Table showing top five genes that were significantly upregulated in the immune class in the MSS samples of COAD cohort. (C) Bubble plot representation showing the correlation between top 5 genes and 13 immune cell or immune response signatures in the MSS samples of COAD cohort. (D) The left panel shows GBP2 expression between 275 CRC specimens and 347 normal adjacent tissues from the The Cancer Genome Atlas (TCGA). The right panel showed GBP2 expression between MSI-H, MSI-L and MSS and normal tissues, respectively. The data were derived from the public Gene Expression Profiling Interactive Analysis2 (GEPIA2) data portal ( http://gepia2.cancer-pku.cn ). (E) Box plots showing expression of GBP2 between immune class, non-immune class and dMMR/MSI samples in the six cohorts. (F) Box plots showing expression of GBP2 between four CMS subtypes in the GSE39582 cohort and COAD cohort. (G) Table showing significant functional gene sets enriched in the MSS samples of COAD cohort using gene set enrichment analysis (GSEA). (H) GO analysis based on the top 200 differential expressed genes that were screened between high vs low GBP2 expression separated by median expression of GBP2 in the MSS samples of COAD cohort. (I) Survival analysis showing the relationship between GBP2 levels and the OS and RFS of samples in GSE39582, COAD and TMA cohorts. (J) The distribution of metastatic or primary CRC tumors in GBP2 high and GBP2 low expression groups in the GSE131418 dataset. *P<0.05, ***p<0.001, ****p<0.0001 vs control group. COAD, colon adenocarcinoma; CRC, colorectal cancer; GBP2, guanylate binding protein 2; GO, gene ontology; MSI-H, high frequency microsatellite instability; MSI-L, low frequency microsatellite instability; MSS, microsatellite stability; ns, not significant; TMA, tissue microarray.

Article Snippet: Two human MSS CRC cell lines (HT29 and SW480) and a murine MSS CRC cell line (CT26) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: RNA Binding Assay, Expressing, Derivative Assay, Gene Expression, Functional Assay, Control, Binding Assay, Microarray

Journal: Journal for Immunotherapy of Cancer

Article Title: Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target

doi: 10.1136/jitc-2021-004302

Figure Lengend Snippet: High GBP2 expression is associated with CD8 +T cell infiltration by bioinformatic approaches. (A) t-Distributed stochastic neighbor embedding (t-SNE) plot from eight MSS CRC patients. (B) Violin plot of GBP2 expression in eight MSS CRC patients. (C) t-SNE plot from two lowest GBP2 expression group (left) and highest GBP2 expression patients (right). (D) The correlation between GBP2 and CD8A in the MSS samples of GSE39582 cohort. (E) Bubble plot representation showing the correlation between GBP2 expression and 28 immune cells in the MSS samples of six cohort. (F) Differences in the various steps of the cancer immunity cycle between high-GBP2 and low-GBP2 groups in the MSS samples of GSE39582 cohort. ****P<0.0001 vs control group. CRC, colorectal cancer; GBP2, guanylate binding protein 2; MSS, microsatellite stability; FDA, Food and Drug Administration; MDSC, myeloid-derived suppressor cell.

Article Snippet: Two human MSS CRC cell lines (HT29 and SW480) and a murine MSS CRC cell line (CT26) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Control, Binding Assay, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target

doi: 10.1136/jitc-2021-004302

Figure Lengend Snippet: Verification of high GBP2 expression is associated with CD8 +T cell infiltration. (A) Expression of GBP2, CD8 and PD-L1 in the TMA cohort of 62 MSS CRC were detected using immunofluorescence. Representative costaining images of GBP2, PD-L1, and CD8 in the high and low GBP2 expression. Scale bars: 200 µm. (B) Box plots showing the positive percent of CD8 and PD-L1 between the high and low GBP2 expression group of the MSS samples of TMA cohort. (C) Correlation between the GBP2 positive percent and CD8 positive per cent detected using immunofluorescence. (D) Correlation between the GBP2 positive percent and PD-L1 positive per cent detected using immunofluorescence. (E) TMA were divided into two immune (>5%) and non-immune class (≤5%) based on the positive percent of CD8 +T cells. Box plots showing the positive percent of GBP2 between the immune and non-immune class of the MSS samples of TMA cohort. (F) (Bottom left) Correlations between GBP2 and 13 immune cell or immune response signatures. (Top right) Correlations between GBP2 and the steps of the cancer immunity cycle. (G) Survival analysis showing the relationship between GBP2 levels and the OS of patients in the MSS samples of TMA. CRC, colorectal cancer; GBP2, guanylate binding protein 2; MSS, microsatellite stability; OS, overall survival; TMA, tissue microarray.

Article Snippet: Two human MSS CRC cell lines (HT29 and SW480) and a murine MSS CRC cell line (CT26) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Immunofluorescence, Binding Assay, Microarray

Journal: Journal for Immunotherapy of Cancer

Article Title: Subtyping of microsatellite stability colorectal cancer reveals guanylate binding protein 2 (GBP2) as a potential immunotherapeutic target

doi: 10.1136/jitc-2021-004302

Figure Lengend Snippet: High expression of GBP2 is essential for responsiveness to PD-1 blockade in pMMR/MSS CRC. (A) GSEA plots of reactome PD-1 signaling and showing positively correlation with higher expression of GBP2 in the MSS samples of GSE39582. (B) Box plots showing expression of ICB response-related signatures between high and low GBP2 group in the GSE39582. (C) Submap analysis demonstrates that the high GBP2 group in the MSS samples of GS39582 cohort are nearly identical to the PD1-response (PD1-R) group defined in the melanoma cohort. (D) The correlation between GBP2 and PD-L1 in the MSS samples of GSE39582. (E) Transfection efficiency of two GBP2 knockout CT26 cells. (F) Time-course evaluation of NSG tumor volumes measured every 6 days. (G) Schematic diagram showing the grouping and treatment plan of the in vivo study: BALB/c mice were inoculated with 10 6 Vec or GBP2 KO CT26 cells and received PD-1 mAb treatment or IgG2b control at the indicated time points. (H) (left) Time-course evaluation of BALB/c tumor volumes measured every 5 days; (right) Tumor inhibition rate for GBP2 KO cells relative to Vec cells under anti-PD-1 treatment, respectively. (I) Representative dot blot of flow cytometric analysis of CD45 +CD3+CD8+cells. (J) The quantitative percentage of CD8 between Vec and GBP2 knockout tumors were shown. (K) Representative images and statistical quantitation of the FACS analysis of the percentage of IFN-γ+CD8+ and TNF-α+CD8+TILs from Vec and GBP2 KO xenografts. (L) Representative photographs show the intratumoral expression of CD8A, MHC class I, CXCL10, and CXCL11 in samples with high GBP2 and in samples with low GBP2 in the same fields, on serial sections in CRC. Scale bars: 100 µm. The corresponding quantitative results were also shown. (M) A schematic show that the upregulation of GBP2 expression promotes the expression of CXCL10/11 and APM by competitively binding p-STAT1 with SHP1, thereby enhancing the anti-PD-1 response in the ‘immune hot’ MSS CRC. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs control group. APM, antigen processing and presentation machinery; CRC, colorectal cancer; ES, enrichment score; FDR, false discovery rate; GBP2, guanylate binding protein 2; ICB, immune checkpoint blockade; MHC, immunohistochemistry; MSS, microsatellite stability; NES, normalized enrichment score; STAT1, signal transducer and transcription activator 1.

Article Snippet: Two human MSS CRC cell lines (HT29 and SW480) and a murine MSS CRC cell line (CT26) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Expressing, Transfection, Knock-Out, In Vivo, Control, Inhibition, Dot Blot, Quantitation Assay, Binding Assay, Immunohistochemistry