Journal: Nature Microbiology

Article Title: Peptostreptococcus anaerobius mediates anti-PD1 therapy resistance and exacerbates colorectal cancer via myeloid-derived suppressor cells in mice

doi: 10.1038/s41564-024-01695-w

Figure Lengend Snippet: a , Representative images of MDSCs that migrated to the lower chamber and quantification of MDSC migration towards CRC-cell-conditioned medium pre-incubated with vehicle or P. anaerobius ( n = 3 biologically independent samples). Scale bars, 100 μm. b , Cytokine array analysis (36 cytokines) of conditioned medium from Caco-2 cells treated with P. anaerobius or vehicle (left). Fold change in cytokine levels after P. anaerobius treatment (right). Red box, significantly changed cytokines; NC, negative control. c , Levels of CXCL1 secreted from Caco-2 and MC38 cells treated with vehicle or P. anaerobius (left). Levels of serum CXCL1 and mRNA expression of CXCL1 in the colon of Apc min/+ mice (right). d , Integrin α 2 β 1 knockdown in Caco-2 cells abrogated P. anaerobius -induced CXCL1 secretion (left) and migration of MDSCs (right). e , RGDS peptide treatment of MC38 cells abrogated P. anaerobius -induced CXCL1 secretion (left) and migration of MDSCs (right). f , Treatment with JSH-23 abolished P. anaerobius -induced CXCL1 secretion (left) and MDSCs migration (right). c – f , n = 3 biologically independent samples. g – j , AOM-induced CRC mice were treated with RGDS. g , Tumour load of the mice; n = 8 (PBS), 11 (PA), 7 (PBS + RGDS) and 10 (PA + RGDS) mice. h , Serum levels of CXCL1; n = 5 (PBS and PA) and 4 (PBS + RGDS and PA + RGDS). i , Percentage of MDSCs in the colonic lamina propria of the mice (left). Immunofluorescence images (middle) and quantification of CD11b + Gr-1 + cells in mouse tumours (right). Scale bars, 25 μm. j , Mean fluorescence intensity (MFI) of IFN-γ + CD8 + and IFN-γ + CD4 + T cells in colonic lamina propria. i , j , n = 7 (PBS and PBS + RGDS) and 9 (PA and PA + RGDS) mice. k – m , MC38 allograft model mice were treated with RGDS peptide and anti-PD1. k , Tumour growth curves (left) and tumour weight (right); n = 7 (PBS + anti-PD1) and 8 (all other groups) mice. l , CXCL1 levels in tumour tissue lysates; n = 5 (PBS + IgG, PBS + anti-PD1 and PBS + anti-PD1 + RGDS), 8 (PA + IgG), 7 (PA + anti-PD1) and 3 (PA + anti-PD1 + RGDS) mice. m , Percentage of MDSCs (left) and IFN-γ + CD8 + T cells (right) in tumours; n = 7 (PBS + anti-PD1) and 8 (all other groups) mice. a , c – m , Data are the mean ± s.e.m. P values were calculated using a one-way ANOVA, followed by Tukey’s post-hoc test ( a , d – j ); an unpaired two-tailed Student’s t -test ( c ); a one-way ANOVA, followed by Fisher’s least significant difference test ( k – m ) or a two-way ANOVA, followed by Bonferroni’s post-hoc test ( k ).

Article Snippet: For the inhibitor-treatment experiments, cells were pretreated with 30 μM NF-κB inhibitor JSH-23 (MCE) or 100 μM integrin α 2 β 1 inhibitor RGDS peptide (MCE) for 24 h and then treated with P. anaerobius .

Techniques: Migration, Incubation, Negative Control, Expressing, Knockdown, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: Nature Microbiology

Article Title: Peptostreptococcus anaerobius mediates anti-PD1 therapy resistance and exacerbates colorectal cancer via myeloid-derived suppressor cells in mice

doi: 10.1038/s41564-024-01695-w

Figure Lengend Snippet: a . Validation of integrin α 2/ β 1 knock down efficiency by western blot in HCT116 and Caco-2 cells (up) and Integrin α 2 β 1 knockdown in HCT116 cells abrogated P. anaerobius-induced CXCL1 secretion and migration of MDSCs (down). b . Antibody blocking of integrin α 2/ β 1 suppressed P. anaerobius -induced NF- κ B activation in HCT116 and MC38 cells (up) and RGDS peptide treatment in HCT116 cells abrogated P. anaerobius -induced CXCL1 secretion and migration of MDSCs (down). a-b n = 3 biologically independent samples. c . P. anaerobius promoted the nuclear translocation of NF-κB p65 and phospho-p65 in CRC cells. d . Induction of NF-κB activation by P. anaerobius was abolished by JSH-23 in HCT116, Caco-2 and MC38 cells. Three independent experiments were repeated with similar results in a–d. e . Treatment with JSH-23 abolished P. anaerobius -induced CXCL1 secretion (left) and MDSCs migration (right) in conditioned medium from HCT116 cell (n = 3 biologically independent samples). f . Schematic diagram of RGDS treatment in mice. RGDS abrogated P. anaerobius -induced colorectal tumorigenesis. g . Representative flow plots of IFNγ + CD8 + T cells and IFNγ + CD4 + T cells in colonic LP cells. h . Schematic diagram of MC38 allograft model. RGDS peptide reversed P. anaerobius -mediated anti-PD1 resistance in MC38 allografts. Representative images of tumours after sacrificing. i . Percentage of G-MDSCs in tumours were determined by flow cytometry analysis. 6-8 mice were used in each group including IgG (n = 8), a-PD1 (n = 6), a-PD1 + RGDS (n = 7), PA+ IgG (n = 8), PA+ a-PD1 (n = 8), PA+ a-PD1 + RGDS (n = 7). j . Representative tumour flow plots of IFNγ + CD8 + T cells in tumours. k . Integrin α 2/ β 1 blockade abolished the effect of P. anaerobius on NF- κ B pathway in MC38 allograft tumours (n = 4 biologically independent samples). Data are presented as mean ± SEM. P values were calculated by one-way ANOVA followed by Tukey’s post-hoc test ( e, i ).

Article Snippet: For the inhibitor-treatment experiments, cells were pretreated with 30 μM NF-κB inhibitor JSH-23 (MCE) or 100 μM integrin α 2 β 1 inhibitor RGDS peptide (MCE) for 24 h and then treated with P. anaerobius .

Techniques: Biomarker Discovery, Knockdown, Western Blot, Migration, Blocking Assay, Activation Assay, Translocation Assay, Flow Cytometry

Journal: Cell Adhesion & Migration

Article Title: Inhibition of Integrin α v β 3 -FAK-MAPK signaling constrains the invasion of T-ALL cells

doi: 10.1080/19336918.2023.2191913

Figure Lengend Snippet: The cell invasion of Jurkat cells with in vitro integrin β3 inhibition. (a) The transwell analysis of invasion of Jurkat cells treated with cyclo(rgdyk) in concentration ladder of 0, 0.2, 0.4, 0.6, 0.8, 1.0 mmol/L after 24 hours. The migrated cells were stained with crystal violet. (b) Column graph of invasive Jurkat cells treated with cyclo(rgdyk) in concentration ladder of 0, 0.2, 0.4, 0.6, 0.8, 1.0 mmol/L after 24 hours detected by transwell analysis. ***P<.01, ***P<.001. (c) The transwell analysis of invasion of control Jurkat cells(ctrl) and those treated with integrin αvβ3 specific antibodies(anti-ITGαvβ3). The migrated cells were stained with crystal violet. (d) Column graph of invasion of control Jurkat cells(ctrl) and those treated with integrin αvβ3 specific antibodies(anti-ITGαvβ3) detected by transwell analysis. **P<.001.

Article Snippet: The specific integrin inhibitor cyclo(Arg-Gly-Asp-d-Tyr-Lys) peptides (cyclo(RGDyK)(S7844; Selleck Chemicals) was used for disturbing the function of integrin.

Techniques: In Vitro, Inhibition, Concentration Assay, Staining, Control

Journal: Cell Adhesion & Migration

Article Title: Inhibition of Integrin α v β 3 -FAK-MAPK signaling constrains the invasion of T-ALL cells

doi: 10.1080/19336918.2023.2191913

Figure Lengend Snippet: Interference of ITGB3 expression decreased the invasiveness of Jurkat cells and Molt-4 cells.(a) the expression of ITGB3 mRNA in common Jurkat cells (ctrl), cells infected by scramble control RNAi virus (sh-NC) and ITGB3 RNAi virus interfered cells (sh-ITGB3). The relative expression level was presented as the ratio of integrin β3 and β-actin, which was applied as endogenous control. ***P<.001, ***P<.01. ( b ) The expression of ITGB3 mRNA in common MOLT-4 cells (ctrl), blank control cells infected by scramble control RNAi virus (sh-NC) and ITGB3 RNAi virus interfered cells (sh-ITGB3). The expression was **P<.001, ***P<.05. (c) The expression of integrin β3 in Jurkat(ctrl), sh-NC and sh-ITGB3 cells detected by Western blot. (d) The expression of integrin β3 in MOLT-4 (ctrl), sh-NC and sh-ITGB3 cells detected by Western blot. The expression of β-actin was used as internal control. (e) The migratory cell counts of Jurkat (ctrl), sh-NC and sh-ITGB3 cells in transwell assays. (f) The column graph of migratory cell counts of Jurkat, BC and shITGB3 cells. ***P<.001. (g) The migratory cell counts of MOLT-4 (ctrl), sh-NC and sh-ITGB3 cells in transwell assays. (h) The column graph of migratory cell counts of MOLT-4 (ctrl), sh-NC and sh-ITGB3 cells. ***P<.001.

Article Snippet: The specific integrin inhibitor cyclo(Arg-Gly-Asp-d-Tyr-Lys) peptides (cyclo(RGDyK)(S7844; Selleck Chemicals) was used for disturbing the function of integrin.

Techniques: Expressing, Infection, Control, Virus, Western Blot

Journal: Cell Adhesion & Migration

Article Title: Inhibition of Integrin α v β 3 -FAK-MAPK signaling constrains the invasion of T-ALL cells

doi: 10.1080/19336918.2023.2191913

Figure Lengend Snippet: Integrin αV not integrin α2b interact with integrin β3 to regulate the migration of T-ALL cells. (a) The transwell assays for T-ALL cell lines, Jurkat and Molt-4, treated with solution control (ctrl), anti-integrin αvβ3 antibodies (anti-itgαvβ3) and anti-integrin α2bβ3 antibodies (anti- itgα2bβ3), respectively. (b, c) The statistical comparison of migrated T-ALL cell count (B: Jurkat, C: Molt-4) between three groups in transwell assays in (A). ***P<.001. (d) Western blot for the immunoprecipitation of α chain integrin interact with integrin β3. IgG and input represent the negative and positive control, respectively.

Article Snippet: The specific integrin inhibitor cyclo(Arg-Gly-Asp-d-Tyr-Lys) peptides (cyclo(RGDyK)(S7844; Selleck Chemicals) was used for disturbing the function of integrin.

Techniques: Migration, Control, Comparison, Cell Counting, Western Blot, Immunoprecipitation, Positive Control

Journal: Soft matter

Article Title: Characterization of the crosslinking kinetics of multi-arm poly(ethylene glycol) hydrogels formed via Michael-type addition

doi: 10.1039/c5sm02668g

Figure Lengend Snippet: This schematic shows the crosslinking kinetics between (A) 4-arm PEG and (B) 8-arm PEG that are modified with integrin binding peptides, RGD, and trifunctional protease sensitive crosslinking peptide, YKNR, through Michael-type addition. Intermolecular reactions and the presence of a third binding site allowed continuation of the network formation. However, bioactive modification with integrin binding peptides, RGD, affected crosslinking kinetics by binding to the reactive sites on the PEG molecules, preventing network growth at those binding sites. L-Cysteine hydrochloride monohydrate (L-Cys) was substituted on a molar basis as a biologically inactive surrogate molecule for RGD.

Article Snippet: PEG hydrogels were modified with the integrin binding peptide G C GYG RGD SPG (RGD) (1067.10 g mol −1 , GenScript) to allow attachment of the encapsulated cells.

Techniques: Modification, Binding Assay