b16 melanoma  (ATCC)

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ p < 0.05.

Article Snippet: B16 melanoma, MC38 colon cancer, and 4T1 breast cancer cell lines were procured from ATCC.

Techniques: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: B16 melanoma, MC38 colon cancer, and 4T1 breast cancer cell lines were procured from ATCC.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: BOLT treatment induces ferroptosis in tumor cells. (A) RNA was extracted from Control and BOLT-treated tumors, and RNA sequencing (RNAseq) was performed to identify differentially expressed genes. (B) KEGG pathway analysis was conducted to assess the biological functions of the differentially expressed genes. (C) Heatmap displaying the differential expression of ferroptosis-related genes in BOLT-treated versus Control cells. (D) qRT-PCR analysis showing dose-dependent downregulation of Nrf2 in BOLT-treated cells. (E) qRT-PCR analysis of Duox1 expression in B16 cells following BOLT treatment. (F) Transmission electron microscopy (TEM) images showing mitochondrial shrinkage, increased membrane density, and loss of cristae in BOLT-treated cells. (G) Heatmap showing the dysregulated genes involved in ROS-chemical carcinogenesis in B16 cells treated with BOLT. (H) Flow cytometry analysis revealing reactive oxygen species (ROS) production in B16 cells treated with BOLT (0.25 μg/mL) compared to Control. (I) Histogram overlays and bar graph confirm elevated bodipy levels in BOLT-treated cells versus Control. (J) Annexin V/PI staining shows no significant apoptosis in B16 cells following BOLT treatment. (K) Western blot analysis showing the expression of genes involved in downregulating ferroptosis (SLC7A11, FACL4, and GPX4) in BOLT-treated B16 cells. Student t-test was performed for comparison between 2 groups. Two-way ANOVA was used for multiple comparisons. In-vitro experiments were performed in triplicate. Data are mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: B16 melanoma, MC38 colon cancer, and 4T1 breast cancer cell lines were procured from ATCC.

Techniques: Control, RNA Sequencing, RNA sequencing, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Transmission Assay, Electron Microscopy, Membrane, Flow Cytometry, Staining, Western Blot, Comparison, In Vitro

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: B16 melanoma, MC38 colon cancer, and 4T1 breast cancer cell lines were procured from ATCC.

Techniques: Injection, Control, Flow Cytometry

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Physicochemical properties of BOLT, and BOLT reduces the growth of tumor cells. (A) Schematic of surface double-layer formation and ion release. (B) Negative zeta potential (−1.365 mV) and high conductivity (1.334 mS/cm), confirming colloidal stability and ion release. (C) Uniform particle size (∼1478 nm) across batches. (D) Interfacial pH buffering in PBS. (E) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with 6.0 pH and treated with various doses of BOLT. RT-qPCR was performed to determine the expression of Gpcr68 at various BOLT doses in activated T cells at acidic pH. (F) Anti-CD3 and anti-CD28 activated CD4 + T cells were treated with different doses of BOLT to determine the protein expression of GPCR68 using Western blot. (G-J) CCK8 assay was performed to analyze the effect of various pH on B16, MC38, 143B, and MG63 cell proliferation. (K-L) Effect of various doses of BOLT on the B16 and K7M2 cell growth to determine the IC-50 of BOLT. Error bars represent mean ± SEM. ∗∗ p < 0.01 and ∗ p < 0.05.

Article Snippet: Murine tumor cell lines B16 melanoma (RRID: CVCL_0159), MC38 colon cancer (RRID: CVCL_B288), and 4T1 (RRID: CRL_2539) were purchased from the ATCC and cultured in RPMI 1640 medium (Gibco) or DMEM medium (Gibco) with 10% FBS as well as 1% penicillin/streptomycin.

Techniques: Zeta Potential Analyzer, Isolation, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Murine tumor cell lines B16 melanoma (RRID: CVCL_0159), MC38 colon cancer (RRID: CVCL_B288), and 4T1 (RRID: CRL_2539) were purchased from the ATCC and cultured in RPMI 1640 medium (Gibco) or DMEM medium (Gibco) with 10% FBS as well as 1% penicillin/streptomycin.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: BOLT treatment induces ferroptosis in tumor cells. (A) RNA was extracted from Control and BOLT-treated tumors, and RNA sequencing (RNAseq) was performed to identify differentially expressed genes. (B) KEGG pathway analysis was conducted to assess the biological functions of the differentially expressed genes. (C) Heatmap displaying the differential expression of ferroptosis-related genes in BOLT-treated versus Control cells. (D) qRT-PCR analysis showing dose-dependent downregulation of Nrf2 in BOLT-treated cells. (E) qRT-PCR analysis of Duox1 expression in B16 cells following BOLT treatment. (F) Transmission electron microscopy (TEM) images showing mitochondrial shrinkage, increased membrane density, and loss of cristae in BOLT-treated cells. (G) Heatmap showing the dysregulated genes involved in ROS-chemical carcinogenesis in B16 cells treated with BOLT. (H) Flow cytometry analysis revealing reactive oxygen species (ROS) production in B16 cells treated with BOLT (0.25 μg/mL) compared to Control. (I) Histogram overlays and bar graph confirm elevated bodipy levels in BOLT-treated cells versus Control. (J) Annexin V/PI staining shows no significant apoptosis in B16 cells following BOLT treatment. (K) Western blot analysis showing the expression of genes involved in downregulating ferroptosis (SLC7A11, FACL4, and GPX4) in BOLT-treated B16 cells. Student t-test was performed for comparison between 2 groups. Two-way ANOVA was used for multiple comparisons. In-vitro experiments were performed in triplicate. Data are mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Murine tumor cell lines B16 melanoma (RRID: CVCL_0159), MC38 colon cancer (RRID: CVCL_B288), and 4T1 (RRID: CRL_2539) were purchased from the ATCC and cultured in RPMI 1640 medium (Gibco) or DMEM medium (Gibco) with 10% FBS as well as 1% penicillin/streptomycin.

Techniques: Control, RNA Sequencing, RNA sequencing, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Transmission Assay, Electron Microscopy, Membrane, Flow Cytometry, Staining, Western Blot, Comparison, In Vitro

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Murine tumor cell lines B16 melanoma (RRID: CVCL_0159), MC38 colon cancer (RRID: CVCL_B288), and 4T1 (RRID: CRL_2539) were purchased from the ATCC and cultured in RPMI 1640 medium (Gibco) or DMEM medium (Gibco) with 10% FBS as well as 1% penicillin/streptomycin.

Techniques: Injection, Control, Flow Cytometry

Journal: iScience

Article Title: Targeting ADAR1 p150 triggers tumor inhibition and antitumor immunity to overcome immunotherapy resistance

doi: 10.1016/j.isci.2026.115978

Figure Lengend Snippet: p150 depletion is sufficient to inhibit tumor growth inhibition and induce tumor inflammation (A) Schematic of the experimental design using syngeneic transplant models in IFN-β/Luc reporter mice. (B) Tumor growth curves of wild-type versus ADAR1-knockout B16-F10 cells in C57BL/6 mice ( n = 10 mice per group). Data are mean ± SEM. Statistical significance was determined by two-way ANOVA (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (C and D) In vivo bioluminescence imaging (D) and quantification of photon flux (E) from tumors in IFN-β/Luc reporter mice bearing WT or ADAR1-KO B16-F10 tumors ( n = 5 mice per group). Data are mean ± SD. Unpaired t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (E) Flow cytometric analysis of tumor-infiltrating leukocytes in WT and ADAR1-KO B16-F10 tumors ( n = 4 tumors per group from two independent experiments). (F) Cytokine levels of IFN-β and CXCL10 in tumor homogenates measured by MultiPlex ( n = 5 tumors per group). Data are mean ± SD. Unpaired t test (∗∗ p < 0.01, ∗∗∗ p < 0.001). (G and H) Interferon-stimulated genes (ISGs) expression (H) and heatmap of DEGs with corresponding gene enrichment analysis (I) of RNA-seq data from WT and ADAR1-KO B16-F10 cells ( n = 2 biological replicates per group), showing upregulation of ISGs and dsRNA sensing pathways.

Article Snippet: Mouse: B16-F10 (Melanoma) , ATCC , CRL-6475.

Techniques: Inhibition, Knock-Out, In Vivo, Imaging, Multiplex Assay, Expressing, RNA Sequencing

Journal: iScience

Article Title: Targeting ADAR1 p150 triggers tumor inhibition and antitumor immunity to overcome immunotherapy resistance

doi: 10.1016/j.isci.2026.115978

Figure Lengend Snippet: Genetic disruption of the Zα domain inhibits tumor growth and activates anti-tumor immunity (A) Schematic of the B16-F10 cells engineered to express WT ADAR1 p150 or Zα domain mutants (N175S, Y179A). (B) Heatmap of DEGs between WT and Zα (N175S) mutant cells under basal conditions ( n = 3 biological replicates per group). (C) Gene ontology (GO) enrichment analysis of upregulated pathways in Zα mutant cells compared to WT. (D) Heatmap of ISG upregulation in Zα mutant cells compared to WT under PBS and IFNb condition. (E) Colony formation assay of WT and Zα mutant cells with or without IFN-β treatment. Scale bar, 10 mm. (F) Tumor growth curves of syngeneic mice implanted with B16-F10 cells expressing WT, heterozygous N175S, homozygous Y179A, or homozygous N175S ADAR1 p150 ( n = 8 mice per group). two-way ANOVA ([∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001] for mutants vs. WT). (G) Flow cytometric quantification of tumor-infiltrating leukocytes in WT and Zα (N175S) mutant tumors at endpoint ( n = 6 tumors per group). Data are mean ± SD. two-way ANOVA ([∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001] for mutants vs. WT).

Article Snippet: Mouse: B16-F10 (Melanoma) , ATCC , CRL-6475.

Techniques: Disruption, Mutagenesis, Colony Assay, Expressing