Journal: ACS biomaterials science & engineering

Article Title: Titrating polyarginine into nanofibers enhances cyclic-di-nucleotide adjuvanticity in vitro and after sublingual immunization

doi: 10.1021/acsbiomaterials.0c01429

Figure Lengend Snippet: (A) Positively charged PEG-Q11R9 nanofibers were rapidly acquired by dendritic cells in vitro , as compared with PEG-Q11OVA nanofibers or soluble pOVA peptide. TAMRA-labelled peptides or peptide nanofibers were incubated for 1 or 4 hours with murine DC2.4 dendritic cells and the uptake was measured by flow cytometry. *** p < 0.001, 2-way ANOVA with Tukey’s multiple comparisons test, n=6/group in two independent experiments. (B) Co-assembly of PEG-Q11OVA with PEG-Q11R9 promoted presentation of the pOVA epitope in MHC class II molecules, as measured by DOBW reporter T cells, which secrete IL-2 upon encountering DCs with pOVA loaded MHC II. IL-2 concentration in the supernatant was measured by ELISA. Inclusion of STING agonist adjuvants cyclic-di-AMP (c-di-AMP) or cyclic-di-GMP (c-di-GMP) did not alter presentation. **** p < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test, n=3/group. MFI = mean fluorescence intensity.

Article Snippet: Vaccine grade cyclic-di-GMP (Invivogen, vac-nacdg) and cyclic-di-AMP (Invivogen, vac-nacda) adjuvants were mixed with nanofiber solutions at a dose of 10 μg/mouse.

Techniques: Immunopeptidomics, In Vitro, Incubation, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: ACS biomaterials science & engineering

Article Title: Titrating polyarginine into nanofibers enhances cyclic-di-nucleotide adjuvanticity in vitro and after sublingual immunization

doi: 10.1021/acsbiomaterials.0c01429

Figure Lengend Snippet: PEG-Q11R9 nanofibers mixed with cyclic-di-GMP (c-di-GMP) adjuvant led to significant upregulation of the dendritic cell activation markers CD80 (A) and CD86 (B) . Murine DC2.4 dendritic cells were incubated with indicated treatments overnight and activation level was assessed by flow cytometry. **** p < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test, n=2–5/group. Titrating the PEG-Q11R9 content in nanofibers led to a PEG-Q11R9-dose dependent increase in CD80 and CD86 when mixed with (C) c-di-GMP or (D) the related STING agonist adjuvant cyclic di-AMP (c-di-AMP). n=4/group by two independent experiments of n=2/group (C) or n=3/group (D) . To analyze the PEG-Q11R9-dependent increase on CD80 and CD86 levels, the fold-change in MFI was plotted against PEG-Q11R9 nanofiber content and a linear regression was performed (E, F) . ** p < 0.01, *** p < 0.001, linear regression of MFI fold change vs. molar percentage of PEG-Q11R9 in nanofiber. MFI = mean fluorescence intensity.

Article Snippet: Vaccine grade cyclic-di-GMP (Invivogen, vac-nacdg) and cyclic-di-AMP (Invivogen, vac-nacda) adjuvants were mixed with nanofiber solutions at a dose of 10 μg/mouse.

Techniques: Activation Assay, Adjuvant, Incubation, Flow Cytometry, Fluorescence

Journal: ACS biomaterials science & engineering

Article Title: Titrating polyarginine into nanofibers enhances cyclic-di-nucleotide adjuvanticity in vitro and after sublingual immunization

doi: 10.1021/acsbiomaterials.0c01429

Figure Lengend Snippet: Mixing PEG-Q11R9 nanofibers with either cyclic-di-GMP (c-di-GMP) or cyclic-di-AMP (c-di-AMP) led to significant upregulation of the activation markers (A) CD80 and (B) CD86 on murine DC2.4 dendritic cells. By contrast, combining PEG-Q11R9 with a structurally different nucleotide adjuvant, CpG, did not increase DC activation compared with PEG-Q11R9 alone. DCs were incubated overnight with the indicated treatments and activation was measured by flow cytometry. ** p < 0.01, *** p < 0.001, **** p < 0.0001, 1-way ANOVA with Dunnett’s multiple comparisons test vs. PEG-Q11R9 only, n=3/group. MFI = mean fluorescence intensity.

Article Snippet: Vaccine grade cyclic-di-GMP (Invivogen, vac-nacdg) and cyclic-di-AMP (Invivogen, vac-nacda) adjuvants were mixed with nanofiber solutions at a dose of 10 μg/mouse.

Techniques: Activation Assay, Adjuvant, Incubation, Flow Cytometry, Fluorescence

Journal: ACS biomaterials science & engineering

Article Title: Titrating polyarginine into nanofibers enhances cyclic-di-nucleotide adjuvanticity in vitro and after sublingual immunization

doi: 10.1021/acsbiomaterials.0c01429

Figure Lengend Snippet: The synergistic upregulation of CD80 and CD86 on dendritic cells achieved by mixing PEG-Q11R9 and cyclic-di-nucleotide adjuvants is ablated by treatment of DCs with a small-molecule inhibitor of the STING pathway (C-178). Additionally, mixing PEG-Q11R9 with a non-STING activating control, cyclic-di-UMP (c-di-UMP), does not promote the same level of DC activation as cyclic-di-AMP (c-di-AMP) or cyclic-di-GMP (c-di-GMP). (A-D) Representative histograms showing CD80 and CD86 MFI plotted against mode-normalized cell counts. Murine DC2.4 dendritic cells were incubated overnight with the indicated treatments and CD80 and CD86 levels were measured by flow cytometry. STING-inhibited groups were incubated with C-178 for 4 hours prior to treatment with nanofibers and/or adjuvants. (E-F) Quantification of experiments depicted in A-D. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, 2-way ANOVA with Tukey’s multiple comparisons test; stars in “No Inhibitor” grouping indicates differences between “No Inhibitor” groups; stars in “+ STING Inhibitor” groups indicates differences between “No Inhibitor” and “+ STING Inhibitor” for a given treatment condition; n=3/group. MFI = mean fluorescence intensity.

Article Snippet: Vaccine grade cyclic-di-GMP (Invivogen, vac-nacdg) and cyclic-di-AMP (Invivogen, vac-nacda) adjuvants were mixed with nanofiber solutions at a dose of 10 μg/mouse.

Techniques: Adjuvant, Activation Assay, Control, Incubation, Flow Cytometry, Fluorescence

Journal: ACS biomaterials science & engineering

Article Title: Titrating polyarginine into nanofibers enhances cyclic-di-nucleotide adjuvanticity in vitro and after sublingual immunization

doi: 10.1021/acsbiomaterials.0c01429

Figure Lengend Snippet: (A) Hypothesized structure of assembled PEG-Q11R9 nanofibers or non-assembled R9 peptides interacting with cyclic-di-nucleotides. The densities of PEG and R9 ligands on Q11 are reduced for visual clarity. Polyvalent display of R9 ligands on PEG-Q11R9 nanofibers mixed with cyclic-di-GMP (c-di-GMP) or cyclic-di-AMP (c-di-AMP) led to significantly greater upregulation of (B) CD80 and (C) CD86 by dendritic cells than adjuvant mixed with unassembled R9 peptide. Murine DC2.4 dendritic cells were incubated overnight with the indicated treatments and activation was measured by flow cytometry. *** p < 0.001, 1-way ANOVA with Tukey’s multiple comparisons test, n=3/group. MFI = mean fluorescence intensity.

Article Snippet: Vaccine grade cyclic-di-GMP (Invivogen, vac-nacdg) and cyclic-di-AMP (Invivogen, vac-nacda) adjuvants were mixed with nanofiber solutions at a dose of 10 μg/mouse.

Techniques: Activation Assay, Adjuvant, Incubation, Flow Cytometry, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: Combined c-di-AMP treatment and CD47 blockade elicits strong anticancer activity in vivo. A Schematic showing the treatment strategy for the transplanted MC38 tumor model. B Tumor growth curves in each treatment group ( n = 10). C Representative images of tumors on day 15 after engraftment. D Schematic showing the treatment strategy for the transplanted SW620 tumor model. E Tumor growth curves in each treatment group ( n = 8). F Representative images of tumors on day 13 after engraftment. Two-way ANOVA, 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** 0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Activity Assay, In Vivo, Xenograft Assay

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: C-di-AMP enhances macrophage-mediated phagocytosis. A Growth inhibition of SW620 cells in the absence or presence of BMDMs ( n = 3, two-way ANOVA). B-D. Cell removal assays measured by bioluminescence ( n = 3, two-way ANOVA). B Co-culture of SW620 cells and BMDMs treated with c-di-AMP. C SW620 cells treated with c-di-AMP. D BMDMs treated with c-di-AMP. E and F . Cell removal assays of CT26 and MC38 cells following c-di-AMP treatment of BMDMs ( n = 3, two-way ANOVA). * 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** 0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Inhibition, Co-culture Assay

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: C-di-AMP enhances CD47 blockade-induced phagocytosis. A , B Phagocytosis was assessed via flow cytometry under conditions of 0 µM (Ctrl) or 10 µM c-di-AMP ( n = 3; two-way ANOVA); C . Representative fluorescence microscopy images of phagocytosis assays. BMDMs are labelled in red; SW620 cells in green. Arrows indicate cancer cells undergoing phagocytosis by macrophages (double colors). D Percentage of phagocytized cells ( n = 3; two-way ANOVA). * 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** 0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Flow Cytometry, Fluorescence, Microscopy, Immunofluorescence, FACS

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: C-di-AMP facilitates the M1 polarization of macrophages. A , B Representative FACS plots and mean fluorescent intensity (MFI) of the M1 marker MHCII ( A ) and the M2 marker CD206 ( B ) expression on BMDMs ( n = 3, t-test). C Representative IHC images and CD86- (top), iNOS- (middle), and CD206-positive expression areas (bottom). ( n = 3; one-way ANOVA). * 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; ***0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Marker, Expressing, FACS, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: C-di-AMP promotes phagocytosis by activating the NF-κB signaling pathway. A Activation of the STING signaling pathway assessed using an ISRE reporter system in RAW 264.7 macrophages treated with DMXAA (265µM) or c-di-AMP (10 µM) ( n = 3, t-test). B Activation of the NF-κB signaling pathway measured using an NF-κB reporter system in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS; 100 ng/mL) or c-di-AMP (10 µM) ( n = 3, t-test). C Western blot analysis showing expression levels of p-p65, p65, and p-IκBα in BMDMs treated with c-di-AMP (10 µM). D, E. Phagocytosis was quantified via bioluminescence in the presence or absence of 10 µM c-di-AMP, 500 nM TPCA1 ( D ), or 500 nM C-176 ( E ) ( n = 3; two-way ANOVA). * 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** 0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Activation Assay, Western Blot, Analysis, Expressing, Luciferase, Phagocytosis Assay

Journal: Cell Communication and Signaling : CCS

Article Title: C-di-AMP sensitizes colorectal cancer to CD47 antibodies by activating NF-κB to promote macrophage phagocytosis

doi: 10.1186/s12964-026-02703-8

Figure Lengend Snippet: C-di-AMP enhances macrophage adhesion and phagocytic capacity by upregulating CD11b/CD18 expression. A KEGG enrichment analysis of RNA-seq data reveals differentially enriched pathways in PBS-treated (control) and 10 µM c-di-AMP-treated BMDMs. B Relative mRNA expression was measured using RT-PCR in an independent experiment ( n = 3, t-test). C , D Representative FACS plots showing the expression of CD11b and CD18 on BMDMs treated with c-di-AMP or PBS (control) ( n = 3, t-test). E Representative IHC images showing the expression of F4/80 + in tumor tissues, F4/80-positive area (right) ( n = 4, one-way ANOVA). F Quantification of adhesion between BMDMs and SW620 cells assessed via bioluminescence. Anti-CD11b (5 µg/mL) or anti-CD18 (8 µg/mL) were added to assess their effect ( n = 4, one-way ANOVA). G Cell removal assays were conducted using SW620 cells co-cultured with c-di-AMP-treated BMDMs in the presence or absence of anti-CD11b (5 µg/mL) or anti-CD18 (8 µg/mL) antibodies ( n = 4, one-way ANOVA). * 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** 0.0001 ≤ P < 0.001; **** P < 0.0001

Article Snippet: Once tumors were established, the mice received weekly intratumoral injections of either 1 μg of c-di-AMP or 50 μg of magrolimab (Hu5F9-G4, Targetmol).

Techniques: Expressing, Analysis, RNA Sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Cell Culture, FACS, Immunohistochemistry