Journal: bioRxiv

Article Title: Functional and sensitivity profiling of the KIT Mutation Landscape in Melanoma

doi: 10.64898/2026.02.18.706482

Figure Lengend Snippet: KIT mutations shows differential responses towards common KIT inhibitors in vitro. (A) Western Blot analysis revealed changes in cell growth and proliferation pathways upon expression of KIT mutants in MeWO melanoma cells. (B) Upper panel: Monitoring relative growth of melanoma cell spheroids expressing KIT mutants using high-content microscopy. Lower panel: Representative image depicting spheroid size after 8 days of growth in 3D environment. (C) Western Blot demonstrating the expression of KIT in Ba/F3 cells. (D) Assessment of relative growth of Ba/F3 cells expressing different KIT mutants following withdrawal of IL-3 from the culture medium, measured using cell-titre glo assay. (E) Heatmap shows the relative IC50 of Ba/F3 cell with KIT mutants against KIT inhibitors. (F) Violin plot illustrating the IC50 values of MeWo cells expressing KIT mutants when treated with various KIT inhibitors. Results were obtained from three independent experiments. The horizontal line represents the mean, and statistical analysis was performed using one-way ANOVA.

Article Snippet: Small molecule inhibitors (Imatinib, Sunitinib, Nilotinib, Nintedanib, and Ripretinib) were purchased from MedChemExpress, dissolved in DMSO to a 10 mM stock, and stored at −80°C.

Techniques: In Vitro, Western Blot, Expressing, Microscopy, Glo Assay

Journal: bioRxiv

Article Title: Functional and sensitivity profiling of the KIT Mutation Landscape in Melanoma

doi: 10.64898/2026.02.18.706482

Figure Lengend Snippet: In vivo anti-tumor efficacy of KIT inhibitors against MeWo cells expressing KIT WT/mutants in a mouse xenograft model. (A-C) Response of MeWO-KIT-WT, MeWo-KIT p.L576P, and MeWo-KIT-p.N822K mouse xenograft models to Imatinib, Sunitinib, Nilotinib, and Nintedanib, respectively (n = 3 independent experiments). (D-F) Response of MeWO-KIT-WT, MeWo-KIT p.L576P, and MeWo-KIT-p.N822K mouse xenograft models to Ripretinib (n = 3 independent experiments). Fraction of tumor growth represents the change in tumor volume normalized to day 0 of treatment.

Article Snippet: Small molecule inhibitors (Imatinib, Sunitinib, Nilotinib, Nintedanib, and Ripretinib) were purchased from MedChemExpress, dissolved in DMSO to a 10 mM stock, and stored at −80°C.

Techniques: In Vivo, Expressing

Journal: Advanced Science

Article Title: Vitiligo Signature‐Based Drug Screening Identifies Fulvestrant as a Novel Immunotherapy Combination Strategy

doi: 10.1002/advs.202503979

Figure Lengend Snippet: Fulvestrant and Motolimod enhanced the effects of anti‐PDL1 treatment. A) Relative T cell proportion in B16‐F10 cell and B16‐F10‐OVA cell treated with 50 and 500 nM Fulvestrant for 48 h. B) Relative T cell proportion in B16‐F10 cell and B16‐F10‐OVA cell treated with 50 and 500 nM Motolimod for 48 h. C,F,I,L,O,R) Illustration of animal models. B16‐F10 cells were injected into C57BL mice. Animals were administrated when the volumes of tumors were ≈50mm 3 . 4T1 cells and CT26 cells were injected into Balbc mice. Animals were administrated when the volumes of tumors were ≈50 mm 3 . D,G,J,M,P,S) Mean tumor volume (mm 3 ) of B16‐F10, 4T1, and CT26 during treatment with Fulvestrant (150 mg kg −1 ) and Motolimod (2 mg kg −1 ) or the combination. n=5 tumors. The growth of B16‐F10 tumors, 4T1 tumors, and CT26 tumors were measured by tumor volume; volume (mm 3 ) = [width 2 (mm 2 ) × length (mm)]/2. E,H) Kaplan‐Meier plots demonstrating the association between B16‐F10 tumors and overall survival. K,N,Q,T) Tumor sizes at day 19 (sample‐paired Student's t ‐test). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Error bars depict SEM.

Article Snippet: The small‐molecule inhibitors Fulvestrant (T2146), Cobicistat (T6246), and Motolimod (T6898) were purchased from TargetMol, and were suspended in 10% 2‐hydroxypropyl‐β‐cyclodextri(H108813, Aladdin) and 4% DMSO (PHR1309, Sigma) in water.

Techniques: Injection

Journal: Advanced Science

Article Title: Vitiligo Signature‐Based Drug Screening Identifies Fulvestrant as a Novel Immunotherapy Combination Strategy

doi: 10.1002/advs.202503979

Figure Lengend Snippet: Cytotoxic T cells significantly increased after Fulvestrant perturbation. A) 19 cell clusters were identified by unsupervised clustering. B) 11 cell types were annotated based on established cell markers. C) the differences in proportions of all 11 cell types among control, E2, and E2+F samples. D) Representative images of immunofluorescence (IF) staining showed the distribution of CD8+T cells, CD4+T cells, and GZMB cells in control+anti‐PDL1 and Fulvestrant+anti‐PDL1 treated 4T1 tumors. Scale bar, 20um. E) Statistic analy‐ sis of IF staining results of CD8+ T cells, CD4+ T cells, and GZMB cells in control+anti‐PDL1 and Fulves‐ trant+anti‐PDL1 treated 4T1 tumors. F,G) Representative flow cytometry plots of CD8+ and CD4+ T cells (F) and the percentages of CD8+ and CD4+ T cells within the CD45+ cell population (G) in B16F10 (OVA) xeno‐ graft tumors ( n =6) after four injections of PD‐L1 antibody in different treatment groups. One‐way ANOVA was used to determine statistical significance. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. H) Representative images of IF staining showing the distribution of CD8+ T cells and CD4+ T cells Scale bar, 20um. Control and Fulvestrant treat groups, respectively. I) Representative IF analysis of CD8+ T cells and CD4+ T cells in independent samples.

Article Snippet: The small‐molecule inhibitors Fulvestrant (T2146), Cobicistat (T6246), and Motolimod (T6898) were purchased from TargetMol, and were suspended in 10% 2‐hydroxypropyl‐β‐cyclodextri(H108813, Aladdin) and 4% DMSO (PHR1309, Sigma) in water.

Techniques: Control, Immunofluorescence, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Vitiligo Signature‐Based Drug Screening Identifies Fulvestrant as a Novel Immunotherapy Combination Strategy

doi: 10.1002/advs.202503979

Figure Lengend Snippet: Fulvestrant significantly activated the antigen processing and presentation signaling pathways. A–C) the differences of cell communication strength between the E2 and E2+F group in CXCL, IFN‐II, and MHC‐I signaling pathways. After Fulvestrant perturbation, the cell communication of the above three pathways was increased for the cytotoxic T cells (CD8+ and NK cells) and myoepithelial cells. D) the differences in communication probability between cytotoxic T cells and CD4 T cells or myo‐ epithelial cells.

Article Snippet: The small‐molecule inhibitors Fulvestrant (T2146), Cobicistat (T6246), and Motolimod (T6898) were purchased from TargetMol, and were suspended in 10% 2‐hydroxypropyl‐β‐cyclodextri(H108813, Aladdin) and 4% DMSO (PHR1309, Sigma) in water.

Techniques: Protein-Protein interactions

Journal: Advanced Science

Article Title: Vitiligo Signature‐Based Drug Screening Identifies Fulvestrant as a Novel Immunotherapy Combination Strategy

doi: 10.1002/advs.202503979

Figure Lengend Snippet: Fulvestrant remodels the tumor immune microenvironment by targeting M2‐like macrophages. A,B) Gene ontology (GO) enrichment analysis of downregulated genes from bulk RNA‐seq in Fulvestrant + IgG tumors versus Control + IgG and in Fulvestrant + anti‐PDL1 tumors compared with Control + anti‐PDL1 tumors. Key suppressed pathways include TGFβ signaling, VEGF signaling (blue rectangle), macrophage activation and chemotaxis pathways (red rectangle). C) Volcano plot showing differentially expressed genes in tumors treated with Fulvestrant + IgG compared to Control + IgG. M2 macrophage–associated genes such as Mrc1 , Il10ra , Pdgfra , Gas6 , and Csf1r are significantly downregulated in the Fulvestrant group. D) qPCR validation of Mrc1 , Il10ra , Pdgfra , Gas6 , and Csf1r in tumors treated with Fulvestrant + IgG. n = 4 mice per group; experiments were independently repeated three times. E,F) Immunofluorescence staining quantification of CD206, F4/80, and CD86 cells in tumor tissues. Fulvestrant treatment significantly reduced CD206 macrophages (M2‐like) while having significant effects on CD86 cells. n.s., not significant; ** p < 0.01; *** p <0.001 by unpaired t‐test. G,H) Flow cytometry analysis of bone marrow–derived macrophages (BMDMs) polarized toward the M2 phenotype in vitro in the presence or absence of Fulvestrant. All data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired t ‐test or one‐way ANOVA as appropriate.

Article Snippet: The small‐molecule inhibitors Fulvestrant (T2146), Cobicistat (T6246), and Motolimod (T6898) were purchased from TargetMol, and were suspended in 10% 2‐hydroxypropyl‐β‐cyclodextri(H108813, Aladdin) and 4% DMSO (PHR1309, Sigma) in water.

Techniques: RNA Sequencing, Control, Activation Assay, Chemotaxis Assay, Biomarker Discovery, Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, In Vitro

Journal: bioRxiv

Article Title: Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

doi: 10.1101/2025.10.24.684401

Figure Lengend Snippet: 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: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

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

Journal: bioRxiv

Article Title: Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

doi: 10.1101/2025.10.24.684401

Figure Lengend Snippet: ( A-C ) Ki67 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, the CD44+CD133+ 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 blotting. 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: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

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

Journal: bioRxiv

Article Title: Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

doi: 10.1101/2025.10.24.684401

Figure Lengend Snippet: (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 blotting. (B) The surface PDL1 expression level was measured by FACS with IFN-γ and Niclosamide. ( C ) The cell viability of MC38 when co-cultured with T cells and pre-treated with Fludarabine and Niclosamide were measured by CCK8 assay. ( D ) The sphere forming units induced in MC38 cells was measured with or without Niclosamide. ( E ) The cell population of CD44+/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: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

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

Journal: bioRxiv

Article Title: Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

doi: 10.1101/2025.10.24.684401

Figure Lengend Snippet: (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 hypoxia condition. ( D ) The Cell viability of MC38 cells treated with different dose of Fludarabine or Niclosamide were measured by CCK8 assay, under normoxia or hypoxia condition. The results are expressed as the mean ± SEM of measurements from 4 ∼ 6 animals per group, and each dot represents a measurement from one animal. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: We evaluated small molecule STAT1 inhibitor Fludarabine (F-ara-A, NSC 118218) (MedChemExpress, Cat. No.: HY-B0069) and STAT3 inhibitor Niclosamide (BAY2353, MedChemExpress, Cat. No.: HY-B0497).

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