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: Molecular Therapy Oncology

Article Title: Oncolytic virotherapy mobilizes tumor-resident, granzyme B-producing bystander CD4 + T cells to inhibit systemic microbial infection

doi: 10.1016/j.omton.2026.201187

Figure Lengend Snippet: Dual protection against tumor and pathogen infection by the OV-BYTE strategy (A) Schematic of the experimental design for (B–D). C57BL/6 mice were infected with LCMV Armstrong and engrafted with MC38 cells on day 60 post-infection. On days 7–12 after tumor engraftment, recipients were daily administered PBS, NDV-WT, or NDV-GP daily via the intratumoral route. On day 15 after tumor engraftment, recipients were infected with either LM-GP 61-80 or IAV-GP 61-80 at an LD 50 dose. (B) Tumor growth curve of MC38 tumor-bearing mice intratumorally treated with PBS, NDV-WT, or NDV-GP as described in (A). (C and D) Survival curves of LM-GP 61-80 (C) and IAV-GP 61-80 (D) infection in MC38-engrafted mice treated with PBS, NDV-WT, or NDV-GP as described in (A). (E) Schematic of the experimental design for (F–H). C57BL/6 mice were infected with LCMV Armstrong and engrafted with B16F10 cells on day 60 post-infection. On days 7–12 after tumor engraftment, recipients were administered PBS, Ad5-WT, or Ad5-GP daily via the intratumoral route. On day 15 after tumor engraftment, recipients were infected with either LM-GP 61-80 or IAV-GP 61-80 at an LD 50 dose. (F) Tumor growth curve of B16F10 tumor-bearing mice intratumorally treated with PBS, Ad5-WT, or Ad5-GP as described in (E). (G and H) Survival curves of LM-GP 61-80 (G) and IAV-GP 61-80 (H) infection in B16F10-engrafted mice treated with PBS, Ad5-WT, or Ad5-GP as described in (E). (I) Schematic of the experimental design. Congenic CD45.1 + SM CD4 + T cells were adoptively transferred into naive C57BL/6 recipients (CD45.2 + ), which were then infected with LCMV Armstrong. On day 60 post-infection, these recipients were engrafted with MC38 cells. On days 7–12, these recipients were administered NDV-GP daily via the intratumoral route. Then, Ly108 hi CD39 lo and Ly108 lo CD39 hi SM CD4 + T cells in the spleens were isolated on day 15 post-tumor engraftment and subsequently transferred into MC38 tumor-bearing mice (no LCMV Armstrong infection) via intravenous injection, along with MC38 tumor-bearing mice receiving no cell transfer as control. One day later, all recipients were infected with LM-GP 61-80 at an LD 50 dose. (J) Survival curve of LM-GP 61-80 infection in groups described in (I). (K) Schematic of the experimental design. WT and Gzmb KO mice were infected with LCMV Armstrong. On day 60 post-infection, splenic LCMV Armstrong-activated CD4 + T MEM cells were harvested and adoptively transferred into another cohort of naive C57BL/6 mice. These recipients, along with control C57BL/6 mice with no CD4 + T MEM cell transfer, were then engrafted with MC38 tumor cells, intratumorally administrated NDV-WT or NDV-GP, and infected with LM-GP 61-80 at the indicated time points. (L) Survival curve of LM-GP 61-80 infection in groups described in (I). All data are representative of at least two independent experiments with at least eight mice per group. Not significant (ns), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by two-way ANOVA (B, F) and log rank (Mantel-Cox) test (C, D, G, H, J, L). Center values and error bars (B, F) indicate mean and SEM.

Article Snippet: B16F10 (CRL-6475) cells were acquired from ATCC.

Techniques: Infection, Isolation, Injection, Control

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