Journal: Military Medical Research

Article Title: Octopus-inspired engineered bacteria with a plug-and-play surface display system achieves enhanced tumor-specific colonization and antitumor immunity

doi: 10.1016/j.mmr.2026.100030

Figure Lengend Snippet: Schematic of the construction and therapeutic mechanism of the octopus-inspired triple-engineered bacteria (OITE strain) . The antitumor attenuated Salmonella typhimurium AISI strain was triple-engineered. (1) ST/SC-RGD×4 “tentacles”-Surface modification: the ST sequence was expressed within the external third loop of OmpA in the AISI strain, resulting in the AISI-ST strain. The ST protein was coexpressed with OmpA (OmpA-ST fusion protein) localized to the bacterial outer membrane. An incubation with SC-RGD×4 protein (SC-RGD×4) led to the formation of AISI-ST/SC-RGD×4 strain (AISI-ST/SC-RGD×4) through ST-SC-mediated covalent conjugation. (2) Dynamic EPS “camouflage”-Immunoactivation engineering: the AISI-H-ST strain was created by introducing the quorum-sensing (QS) promoter pLuxI to control HtrA expression into AISI-ST strain, which specifically increased HtrA-mediated extracellular polysaccharide (EPS) production to amplify bacteria-mediated immune activation. (3) Anti-PD1nanobody (PD1nb) “secretion”-Checkpoint blockade engineering: further programming of AISI-H-ST strain enabled QS-triggered anti-PD1nb secretion, generating the AISI-HP-ST strain. The incubation of AISI-HP-ST strain with SC-RGD×4 protein finally produced the OITE strain (AISI-HP-ST/SC-RGD×4). The intravenously administered OITE strain highly selectively accumulates in tumors through RGD-αvβ3 integrin interactions, with subsequent bacterial proliferation initiating two therapeutic actions: HtrA-mediated immune activation via increasing immune cells infiltration and activation, and secreted PD1nb-based blockade of PD-1/PD-L1 immunosuppressive signaling. OITE. Octopus-inspired, triple-engineered bacterium; AISI. Attenuated Salmonella Δ htrA :: luxI - VNP20009 strains; ST. SpyTag; SC. SpyCatcherΔ; RGD. Arginine-glycine-aspartic acid; HtrA. High-temperature requirement A; PD1. Programmed cell death protein 1; H. HtrA; HP. HtrA and PD1nb; AHL. N-acyl homoserine lactone; MACS. Macrophages; Teffs. Effector T cells; Tregs. Regulatory T cells.

Article Snippet: To prepare the surfaces for the strain adhesion assays, the recombinant mouse integrin αvβ3 (TGAV & ITGB3) heterodimer protein (HY-P700761, MCE, USA) was first diluted to 10 μg/ml in 50 mmol/L carbonate buffer (pH 9.6).

Techniques: Bacteria, Modification, Sequencing, Membrane, Incubation, Conjugation Assay, Control, Expressing, Activation Assay, Produced

Journal: EBioMedicine

Article Title: Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice

doi: 10.1016/j.ebiom.2018.06.004

Figure Lengend Snippet: BC71 targets cell-surface GRP78 but not αvβ5 integrin to induce apoptosis. (a) BC71 induces HUVECs apoptosis in a dose-dependent manner. HUVECs were treated with BC71 (concentration range: 12.5, 25, 50, 100 μM) for 24 h and apoptosis was determined using the cell death ELISA kit (Roche). (b) Anti-GRP78 N-terminal domain antibody blocked the apoptosis function of BC71 in a dose-dependent manner. The apoptosis of the combined treatment with increasing amount of anti-GRP78 N-terminal domain antibody and 100 μM BC71 for 24 h was measured using the Cell Death Detection ELISA. (c) Anti-GRP78 C-terminal domain antibody and (d) anti-αvβ5 antibody did not block BC71 induced apoptosis. For clarity of presentation, data were normalized with that of non-treated (VEGF only) cells, which was set as 1. Data are expressed as mean ± standard error of the mean. The results are representative of at least three independent experiments. Statistical significance was determined using ANOVA. *P < 0.05; **P < 0.01, n ≥ 3.

Article Snippet: Antibodies against integrin αvβ5 (P1F76; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GRP78 (A-10, Santa Cruz Biotechnology) and GRP78 (C-20, Santa Cruz Biotechnology) were used for neutralization.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Therapeutic Advances in Urology

Article Title: The role of chromodomain helicase DNA binding protein 1 (CHD1) in promoting an invasive prostate cancer phenotype

doi: 10.1177/17562872211022462

Figure Lengend Snippet: Expression of extracellular matrix proteins and adhesion molecules in CHD1 KO cells. (a) Graph demonstrating the expression of genes that were altered at least two-fold in the NT2 and CHD1 KO lines, Cr16 and Cr21, compared to RWPE-1. ITGA2 was down-regulated, while ITGA4 is up-regulated in CHD1 KO cells. The integrin ligands, collagen (COL16A1, COL4A2, COL5A1 and COL6A2), FN1, and the laminin component, LAMB3, are downregulated in CHD1 KO cells. The most down-regulated ECM components in the CHD1 KO cells were MMP2, SPARC and VTN. The ECM proteins ITGA4, MMP12, and SELL were up regulated. (b) Plot depicting levels of secreted SPARC protein as detected by ELISA. High levels of SPARC are secreted by RWPE-1 compared to the CHD1 KO lines ( ** p < 0.01). NT2 cell lines secreted higher levels of SPARC than RWPE-1 cell lines ( * p < 0.05). Error bars represent standard deviation. (c) Top panel: Western blot showing levels of SPARC in 30 μg of lysate from RWPE-1, NT2, Cr2, Cr16, and Cr21. SPARC is detectable in RWPE-1 and NT2 but not in the CHD1 KO clones. Bottom panel: loading control. (d) Plot depicting levels of secreted MMP2 protein as detected by ELISA. High levels of MMP2 are secreted by RWPE- 1 compared to the CHD1 KO lines ( ** p < 0.01). NT2 cell lines secreted higher levels of MMP2 than RWPE-1 cell lines ( * p < 0.05). Error bars represent standard deviation. (e) Western blot showing levels of TNC (top panel), VTN (middle panel) and GAPDH (lower panel) in the five cell lines. Cr2 and Cr21 express low levels of TNC compared to RWPE-1 and NT2, while Cr16 expresses TNC levels similar to the parental lines. Cr2 and Cr21 express lower levels of VTN than RWPE-1, while Cr16 expresses similar levels. Bar graphs below show levels of TNC (left) and VTN (right) relative to GAPDH in the five lines. CHD1, chromodomain helicase DNA binding protein 1; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; FN1, fibronectin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ITGA2/4, integrin subunit alpha 2/4; KO, knockout; LAMB3, laminin subunit beta-3 precursor; MMP2/12, matrix metalloproteinase 2/12; NT2, non-target cells; SPARC, secreted protein acidic and rich in cysteine; TNC, tenascin; VTN, vitronectin.

Article Snippet: Primary antibodies used included glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology Inc., 25778), CHD1 (Novus Biologicals, NB100-60411), focal adhesion kinase (FAK) (Cell Signaling Technologies or CST, 3285P), phosphorylated extracellular signal regulated kinase (pErk) (CST, 4370S), total Erk (CST, 9102S), phosphorylated protein kinase B (pAKT) (CST, 4060S), total AKT (CST, 72), SPARC (CST, 8725), pMEK 1/2 (mitogen-extracellular signal-regulated kinase 1/2) (CST, 9154), total MEK 1/2 (CST, 9126), tenascin C (CST, 12221), and vitronectin (CST, 60896).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot, Clone Assay, Control, Binding Assay, Knock-Out

Journal: Cells

Article Title: Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation

doi: 10.3390/cells11020237

Figure Lengend Snippet: YAP1 regulates Wnt5A-induced integrin av and Smad2/3 activation. SKOV-3, OVCAR-3, and CAOV-4 cells were transfected with siRNA Scrambled (Scr) or Wnt5A. ( A ) Integrin αv expression level was assessed by immunoblotting in multicellular aggregates (MCAs) OvCa cells (upper panel) and quantifying bands from three experiments in the lower panel. ( B ) The cells were pre-treated with VP (5 μM) 1 h or treated with rhWnt5A (600 ng/mL) for 14 h alone or VP-pretreated + rhWnt5A then expression levels of integrin αv was determined in MCAs OvCa cells (Left panel), and quantification of bands (right panel). ( C ） The Wnt5A overexpressing OVCAR-3 (C3/OVCAR-3) and SKOV-3 clones (C9/SKOV-3) were treated with CWHM-12 (10 μM) for 24 h. Immunoblot of TGFβ1, pSmad2/3, YAP1 determined in MCAs OvCa cells (Left panel) and quantification of bands (right panels). The lower panel shows the quantification of bands from three independent experiments. GAPDH levels were used as an internal control, and results are expressed as mean ± SD. (D) The immunolocalization of pSmad2/3 and YAP-1 in CWHM-12-treated cells compared to control. Original magnification, ×400. *: p < 0.05, **: p < 0.01 and ***: p < 0.001 compared to untreated control cells (Ctrl) or Scr.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-human pSmad2/3 and mouse monoclonal anti-human Smad2/3 were purchased from (Santa Cruz Biotechnology Inc. Heidelberg, Germany), rabbit polyclonal anti-human TGFβ1, rabbit polyclonal anti-human YAP1, and rabbit polyclonal anti-human integrin αv (Biorbyt Ltd., Cambridge, UK); mouse monoclonal anti-human Wnt5A and rabbit polyclonal anti-human GAPDH rabbit polyclonal anti-human α-SMA (Abcam, Boston, MA, USA).

Techniques: Activation Assay, Transfection, Expressing, Western Blot, Clone Assay, Control

Journal: Cells

Article Title: Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation

doi: 10.3390/cells11020237

Figure Lengend Snippet: Model of Wnt5A involvement in EMT and mesothelial activation and clearance. Wnt5A derived from ovarian cancer cells through cytoskeletal rearrangement could directly or indirectly cause YAP1 phosphorylation, which translocates into the nucleus and induces TGFβ1, integrin αv, Wnt5A, and other EMT markers in this drawing. Integrin αv, in turn, may activate extracellular latent-TGFβ1 and play a pivotal role in the EMT process. In addition, YAP1 may cause retention of Smad2/3 in the nucleus, thereby prolonging their biological activity.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-human pSmad2/3 and mouse monoclonal anti-human Smad2/3 were purchased from (Santa Cruz Biotechnology Inc. Heidelberg, Germany), rabbit polyclonal anti-human TGFβ1, rabbit polyclonal anti-human YAP1, and rabbit polyclonal anti-human integrin αv (Biorbyt Ltd., Cambridge, UK); mouse monoclonal anti-human Wnt5A and rabbit polyclonal anti-human GAPDH rabbit polyclonal anti-human α-SMA (Abcam, Boston, MA, USA).

Techniques: Activation Assay, Derivative Assay, Phospho-proteomics, Activity Assay

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative IHC images of tumour samples from patients with low and high α V expression in tumour cells. Objective: 20×. b Kaplan−Meier curve of OS for stage I treatment-naïve lung cancer patients according to the α V expression by IHC analysis of FFPE tumours. c Kaplan−Meier curve of PFS of PD-1 blockade-treated patients with tumours harbouring low and high expression of α V integrin. d Percentages of anti-PD-(L)1-treated patients displaying α V high tumours among long-responders (LR: PFS > 6 months and OS > 12 months) or fast progressors (FP: defined by “early death” occurring within 12 weeks of treatment initiation). e Representative digital mark-up image of fluorescent IHC of CD8 (green), cytokeratin (turquoise), and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + cell density. Left, the density of CD8 + TIL in α V low and α V high tumours. The numbers of tumours in each group are indicated (* p = 0.046). Scale bar, 2 cm. f Representative digital mark-up image of CD8 + CD103 neg (green), CD8 + CD103 + (orange), CD8 - CD103 + (red), cytokeratin (turquoise) and dapi (blue) staining in α V low and α V high tumour sections. d = CD8 + CD103 + cell density. Left, the density of CD8 + CD103 + (* p = 0.016) and CD8 + CD103 neg ( p = 0.120) cells in tumour regions of α V low and α V high tumours. Scale bar, 2 cm. Each symbol represents an individual cell type from tumour samples; horizontal lines correspond to mean ± standard error of the mean (SEM) ( e , f ). Data were calculated with the log-rank test ( b , c ) and Welch’s two-sided t -test ( e , f ). Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Expressing, Staining

Journal: Nature Communications

Article Title: Integrin-α V -mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

doi: 10.1038/s41467-021-25322-y

Figure Lengend Snippet: a Representative flow cytometry plots (bi-exponential scale) of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells from a lung tumour. Right, percentage of α V expression in EpCAM + E-cadherin + and EpCAM neg E-cadherin neg cells ( n = 18, *** p = 0.0002). b Representative flow cytometry plots of β 6 subunit expression in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells from a tumour sample. Right, expression of β 6 integrin in EpCAM + E-cadherin + α V + and EpCAM neg E-cadherin neg α V + cells ( n = 16), * p = 0.013. c Surface expression of α V , β 6 , and β 8 subunits in the IGR-B2 cell line. d Concentration of total TGF-β in CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells measured by ELISA (*** p = 0.0004). Results are presented as mean ± SEM of six independent experiments. Right, relative luciferase activity in the Mu.1LV cell line transfected with (CAGA)9-Lux reporter plasmid and treated with CM from IGR-B2, IGR-B2T, and IGR-B2T-KO cells, normalized to luciferase activity in Mu.1LV cell treated with CM from IGR-B2. Results are presented as mean ± SEM of six independent experiments (* p = 0.011, **** p < 0.0001). e Expression of α V integrin on IGR-B2T and IGR-B2T-KO cells. An isotype control was included. f Representative photos of the morphology of IGR-B2T and IGR-B2T-KO cells by phase-contrast light microscope from one experiment out of five. Objective: 20×. Each symbol represents the individual cell type from tumour samples ( a , b ); horizontal lines correspond to mean ± SEM ( a , b , d ). Data were calculated with paired Student t -tests ( a , b ) and one-way ANOVA with Tukey’s correction ( d ). ns: non-significant. Source data are provided as a Source Data file.

Article Snippet: The cells were double transfected with integrin α V CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400506) and integrin α V HDR plasmid (Santa Cruz Biotechnology, sc-400506-HDR).

Techniques: Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Light Microscopy