Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: IFNγ up-regulate PD-L1 expression in MC38 cells through IFN-STAT1 signaling. PD-L1 expression on MC38 cell surface was significantly increased in the presence of IFN-γ analyzed by flow cytometry (A) and by immune fluorescent staining (B-C) at 24 hours. (D) When treated with siRNA of STAT1, the up-regulation of PD-L1 was significantly reduced, while siRNA of STAT3 has an opposite effect. (E, F) The small interfering RNA reaches a sufficient knock down of STAT1 and STAT3, as shown by real-time PCR assay. (G-I) The mRNA of STAT1 target genes such as CXCL10 and IL-15 shown similar trend of regulation by IFN-γ as PD-L1. (J) The mRNA of STAT3 target gene, MCL-1, shown totally different regulation under normal condition and under treatment of siRNAs. (K, L) The expression of STAT1/STAT3 normal and phosphorylated proteins, and PD-L1 proteins in MC38 cell lines were measured by Western blotting with different dose of IFN-γ stimulation (K) or after knocking down of STAT1 (siSTAT1) or STAT3 (siSTAT3) (L ). The number on the image indicates the relative abundance of PD-L1 protein (fold of control). The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Flow Cytometry, Staining, Small Interfering RNA, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: IFN-γ modulate cancer cell stemness in MC38 cells indirectly through IFN-STAT3 pathway. (A-C) Ki-67 expression in the MC38 tumor spheres was significantly reduced in the presence of IFN-γ analyzed by flow cytometry and immune fluorescence. (D, E) The tumor sphere forming unit induced in the MC38 cells were also reduced in the presence of IFN-γ (Arrowhead: tumor spheroid. Scale bar: 200μm). (F) When MC38 cells were pre-treated with siRNA of STAT1/STAT3, the SFU show different changes. (G) When MC38 cells were pre-treated with siRNA of STAT1/STAT3, both the CD44 hi CD133- and CD44 hi CD133+ cell population show different trend of regulation by IFN-γ. (H-K) The mRNA of cancer stem cell markers was measured by real-time PCR. (L) The expression of cancer stem cell marker proteins in cell nucleus was measured by Western blot. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, nd, no significant difference.

Article Snippet: STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Marker, Western Blot

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: Niclosamide has inhibition effect on both STAT1 and STAT3, which blocks IFN-γ-induced PD-L1 up-regulation in MC38 cells while also reduces CSCs formation. (A) The expression of STAT1/STAT3 signaling pathway and PD-L1 proteins in MC38 cell lines with Fludarabine and Niclosamide treatment was measured by Western blot. (B) The surface PDL1 expression level was measured by FACS with IFN-γ and Niclosamide treatment. (C) The cell viability of MC38 when co-cultured with T cells and pre-treated with Fludarabine (F-ara-A) or Niclosamide were measured by CCK8 assay. (D) The sphere forming units induced in MC38 cells was measured with or without Niclosamide treatment. (E) The cell population of CD44 hi CD133+ in MC38 treated with Niclosamide was measured by FACS. (F) The expression of cancer stem cell marker proteins in cell nucleus was measured by Western blot. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Expressing, Western Blot, Cell Culture, CCK-8 Assay, Marker

Journal: Frontiers in Immunology

Article Title: Identifying the therapeutic potential of niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the tumor microenvironment

doi: 10.3389/fimmu.2026.1761715

Figure Lengend Snippet: Hypoxia enhances PD-L1 upregulation by IFN-γ, while Niclosamide down-regulates Hif1α under hypoxic conditions. (A, B) The expression level of STAT1/STAT3 signaling pathway and PD-L1 proteins in MC38 cell lines with siRNA (A) or Fludarabine and Niclosamide treatment (B) was measured by Western blot. (C) Cell viability of MC38 cells pre-treated with different dose of IFN-γ and co-cultured with primary T cells were measured by CCK8 assay, under normoxia or hypoxic conditions. (D) The Cell viability of MC38 cells treated with different dose of Fludarabine or Niclosamide were measured by CCK8 assay, under normoxia or hypoxic conditions. The results are expressed as the mean ± SEM of triplicate measurements in each group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Cell Culture, CCK-8 Assay