Journal: bioRxiv

Article Title: Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

doi: 10.64898/2026.01.09.698741

Figure Lengend Snippet: (A) Strategies for modification of GS, including amino acid mutation, covalent modification, cyclization, and multivalency. (B) SPR sensorgrams demonstrating the binding affinity of GS and GR for human integrin α5β1 in a concentration-dependent manner. The equilibrium dissociation constant (K D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C) Molecular docking of GR and GS binding integrin a5b1 protein (grey; PDB: 7NWL) showing the selected possible ligation residues. (D-F) Analysis of integrin α5β1 expression in U87MG cells and tumor tissues by western blot ( D ), and immunohistochemistry ( E-F ) analysis. The band for integrin α5 was approximately 150 kDa. M, marker. C, cell. T, tumor. For immunofluorescence images, green is for integrin α5, red for integrin β1, and blue for nucleus. Scale bar, 50□μm ( E-F ). (G) In vitro cellular uptake of [ 68 Ga]GS and [ 68 Ga]GR in U87MG cell lines. All results are expressed as means ± SEM, as indicated in at least three independent experiments. “*” represents differences compared with the [ 68 Ga]GS. * p < 0.05.

Article Snippet: Recombinant human integrin α5β1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip at 25 □ following a standard amine coupling kit.

Techniques: Modification, Mutagenesis, Binding Assay, Concentration Assay, Ligation, Expressing, Western Blot, Immunohistochemistry, Marker, Immunofluorescence, In Vitro

Journal: bioRxiv

Article Title: Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

doi: 10.64898/2026.01.09.698741

Figure Lengend Snippet: (A) Immunofluorescence images show the expression of integrin α5β1, GFAP, and NEUN of U87MG tumors in orthotopic glioblastoma tumor-bearing mice. Green indicates integrin α5, Red for NEUN, yellow for GFAP, and blue for nucleus. Scale bar, 1 mm. (B-C) Representative fluorescence imaging of mice bearing in orthotopic U87MG tumors ( B ) and ex vivo brains ( C ). Cy5-GS and Cy5-GR were intravenously injected with a dose of 5 mg/kg. (D) Quantification of fluorescence intensity in tumors corresponding to ( C ). (E) Immunohistochemical analysis of integrin α5β1 expression in brain tissue sections from orthotopic glioblastoma tumor-bearing mice. Scale bar, 1 mm. (F-G) Immunofluorescence images of brain tissue sections from orthotopic glioblastoma tumor-bearing mice treated with Cy5-GS ( F ) and Cy5-GR ( G ). Green indicates integrin α5, Red for GS or GR, and blue for nucleus. Scale bar, 1 mm. (H-I) Magnific imaging of brain tissue sections from orthotopic glioblastoma tumor-bearing mice treated with GS-Cy5. Scale bar, 50 μm. All results are expressed as means ± SEM, as indicated in at least three independent experiments. A multiple t-test was used when two groups were compared. The symbol “*” represents differences compared with the Cy5-GS. *** p < 0.001.

Article Snippet: Recombinant human integrin α5β1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip at 25 □ following a standard amine coupling kit.

Techniques: Immunofluorescence, Expressing, Fluorescence, Imaging, Ex Vivo, Injection, Immunohistochemical staining

Journal: Translational Oncology

Article Title: Thermoradiotherapy reverses proliferation, metastasis, and anoikis resistance in glioblastoma through ITGA5-PI3K/AKT axis

doi: 10.1016/j.tranon.2025.102552

Figure Lengend Snippet: ITGA5 is a potential target for thermoradiotherapy and correlates with anoikis-resistance. (A) Differentially expressed proteins of GBM tissues with thermoradiotherapy compared with Ctrl groups identified by the proteomic data. n = 3 samples per group. (B, C) mRNA and protein levels of ITGA5 in GBM tumor tissues and adjacent nontumor tissues. mRNA expression levels of target genes were normalized to those of β-actin. β-actin served as the loading control. n = 3 per group. (D) UMAP projection of single-cell transcriptomes, colored by unsupervised Louvain clustering, identifying 17 distinct clusters. (E) UMAP with cells colored by their experimental group: control (CON) or radiotherapy-treated (RT). (F) Cell type annotation of the clusters based on the expression of canonical marker genes. B, B cell; DC, dendritic cells; NK, natural killer cells; pc, plasmocyte; TAMs, tumor-associated macrophages; treg, regulatory T cells. (G) Restricted expression pattern of ITGA5 in the GBM microenvironment. (H) Radiotherapy alters the cellular composition of the GBM microenvironment. (I) Cell-type-specific expression of ITGA5. (J) Cellular distribution stratified by Itga5 expression. (K) The overall ITGA5 expression in CON and RT groups. (L) KEGG pathway enrichment analyses of GBM tissues with thermoradiotherapy compared with Ctrl groups. n = 3 samples per group. (M) ITGA5, total and phosphorylated PI3K and AKT contents in Ctrl, HT, RT, and HT+RT groups. β-actin served as the loading control. * p < 0.05; ** p < 0.01; *** p < 0.001. Ctrl: control group; HT: hyperthermia group; RT: radiotherapy group; HT+RT: thermoradiotherapy group.

Article Snippet: Primary antibodies, PI3K, pPI3K, N-cadherin, Vimentin, E-cadherin, MMP2, Bax, and Bcl-2 were obtained from Cell Signalling Technology (Danvers, MA, USA), whereas ITGA5, MMP9, AKT, p-AKT, p53, Cyclin D1, PCNA, β-actin, and secondary antibodies were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Expressing, Control, Marker

Journal: Translational Oncology

Article Title: Thermoradiotherapy reverses proliferation, metastasis, and anoikis resistance in glioblastoma through ITGA5-PI3K/AKT axis

doi: 10.1016/j.tranon.2025.102552

Figure Lengend Snippet: ITGA5 promotes the proliferation of GBM. (A) mRNA expression of ITGA5 in U251/ LN229-ITGA5-OE and ITGA5-Ctrl cells. mRNA expression levels of target genes were normalized to those of β-actin. (B) Protein expressing levels of ITGA5, PCNA, P53, and Cyclin D1 in ITGA5-OE and ITGA5-Ctrl GBM cells. β-actin served as the loading control. (C) Statistical results of colony formation in ITGA5-OE and ITGA5-Ctrl groups with or without thermoradiotherapy. (D) Images of EdU assay and statistics of the proliferation efficiency of GBM cells in the indicated groups. Scale bar=50 μm. (E) MRI images of intracranial tumor model in ITGA5-OE and ITGA5-Ctrl groups at 21 days. (F) Representative images of xenograft tumors from mice in ITGA5-OE and ITGA5-Ctrl groups. n = 5 per group. Scale bar=1 cm. (G) Kaplan-Meier overall survival curve of intracranial tumor model in ITGA5-OE and ITGA5-Ctrl groups. (H, I) Tumor weight and volumes of xenograft tumors model mice in ITGA5-OE and ITGA5-Ctrl groups. n = 5 per group. (J) Protein expressing levels of ITGA5, PCNA, P53, and Cyclin D1 in ITGA5-OE and ITGA5-Ctrl GBM tissues. β-actin served as the loading control. (K) mRNA expression of ITGA5 in U251/ LN229-sh-ITGA5#1/2/3 and sh-RNA cells. mRNA expression levels of target genes were normalized to those of β-actin. (L) Protein expressing levels of ITGA5, PCNA, P53, and Cyclin D1 in sh-RNA, sh-ITGA5, and sh-ITGA5+ITGA5 groups. β-actin served as the loading control. (M) Statistical results of colony formation in sh-ITGA5 and sh-RNA groups with or without thermoradiotherapy. (N) Images of EdU assay and statistics of the proliferation efficiency of GBM cells in the indicated groups. Scale bar=50 μm. (O) MRI images of intracranial tumor model in sh-ITGA5 and sh-RNA groups at 21 days. (P) Representative images of xenograft tumors from mice in sh-ITGA5 and sh-RNA groups. n = 5 per group. Scale bar=1 cm. (Q) Kaplan-Meier overall survival curve of intracranial tumor model in sh-ITGA5 and sh-RNA groups. (R, S) Tumor weight and volumes of xenograft tumors model mice in sh-ITGA5 and sh-RNA groups. n = 5 per group. (T) Protein expressing levels of ITGA5, PCNA, P53, and Cyclin D1 in sh-ITGA5 and sh-RNA GBM tissues. β-actin served as the loading control. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ITGA5-Ctrl: control groups for ITGA5-overexpression; ITGA5-OE: ITGA5-overexpression; sh-RNA: control groups for ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down GBM cells complemented 1 μg ITGA5; Ctrl: control groups for thermoradiotherapy; HT+RT: thermoradiotherapy.

Article Snippet: Primary antibodies, PI3K, pPI3K, N-cadherin, Vimentin, E-cadherin, MMP2, Bax, and Bcl-2 were obtained from Cell Signalling Technology (Danvers, MA, USA), whereas ITGA5, MMP9, AKT, p-AKT, p53, Cyclin D1, PCNA, β-actin, and secondary antibodies were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Expressing, Control, EdU Assay, Over Expression

Journal: Translational Oncology

Article Title: Thermoradiotherapy reverses proliferation, metastasis, and anoikis resistance in glioblastoma through ITGA5-PI3K/AKT axis

doi: 10.1016/j.tranon.2025.102552

Figure Lengend Snippet: ITGA5 affects migration, invasion, and EMT of GBM. Transwell assay results for testing the (A) migration and (B) invasion ability of U251/ LN229-ITGA5-OE and ITGA5-Ctrl cells with or without thermoradiotherapy and their statistical results. Scale bar=50 μm. (C, D) Protein levels of migration-, invasion-, and EMT-related molecules (MMP2, MMP9, N—Cadherin, E-Cadherin, and Vimentin) in ITGA5-OE and ITGA5-Ctrl GBM cells and tissues. β-actin served as the loading control. Transwell assay results for testing the (E) migration and (F) invasion ability of U251/ LN229-sh-RNA, sh-ITGA5 and sh-ITGA5+ITGA5 cells with or without thermoradiotherapy and their statistical results. Scale bar=50 μm. Protein levels of migration-, invasion-, and EMT-related molecules (MMP2, MMP9, N—Cadherin, E-Cadherin, and Vimentin) in sh-RNA, sh-ITGA5 and sh-ITGA5+ITGA5 cells (G) or sh-RNA and sh-ITGA5 tissues (H). β-actin served as the loading control. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ITGA5-Ctrl: control groups for ITGA5-overexpression; ITGA5-OE: ITGA5-overexpression; sh-RNA: control groups for ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down GBM cells complemented 1 μg ITGA5; Ctrl: control groups for thermoradiotherapy; HT+RT: thermoradiotherapy.

Article Snippet: Primary antibodies, PI3K, pPI3K, N-cadherin, Vimentin, E-cadherin, MMP2, Bax, and Bcl-2 were obtained from Cell Signalling Technology (Danvers, MA, USA), whereas ITGA5, MMP9, AKT, p-AKT, p53, Cyclin D1, PCNA, β-actin, and secondary antibodies were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Migration, Transwell Assay, Control, Over Expression

Journal: Translational Oncology

Article Title: Thermoradiotherapy reverses proliferation, metastasis, and anoikis resistance in glioblastoma through ITGA5-PI3K/AKT axis

doi: 10.1016/j.tranon.2025.102552

Figure Lengend Snippet: Inhibiting ITGA5 reverses anoikis resistance of GBM. (A) Anoikis assay by Cell Viability/Cytotoxicity Assay of U251/ LN229-ITGA5-OE and ITGA5-Ctrl cells with or without thermoradiotherapy and their statistical results. Scale bar=50 μm. (B) Anoikis assay by flow cytometry of U251/ LN229-ITGA5-OE and ITGA5-Ctrl cells. (C, D) Protein levels of anoikis-related molecules (Bcl-2 and Bax) of ITGA5-OE and ITGA5-Ctrl GBM cells and tissues. β-actin served as the loading control. (E) Anoikis assay by Cell Viability/Cytotoxicity Assay in sh-RNA, sh-ITGA5 and sh-ITGA5+ITGA5 cells with or without thermoradiotherapy and their statistical results. Scale bar=50 μm. (F) Anoikis assay by flow cytometry of U251/ LN229-sh-ITGA5 and sh-RNA cells and their statistical results. Protein levels of anoikis-related molecules (Bcl-2 and Bax) of sh-RNA, sh-ITGA5 and sh-ITGA5+ITGA5 cells (G) or sh-RNA and sh-ITGA5 tissues (H). β-actin served as the loading control. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ITGA5-Ctrl: control groups for ITGA5-overexpression; ITGA5-OE: ITGA5-overexpression; sh-RNA: control groups for ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down GBM cells complemented 1 μg ITGA5; Ctrl: control groups for thermoradiotherapy; HT+RT: thermoradiotherapy.

Article Snippet: Primary antibodies, PI3K, pPI3K, N-cadherin, Vimentin, E-cadherin, MMP2, Bax, and Bcl-2 were obtained from Cell Signalling Technology (Danvers, MA, USA), whereas ITGA5, MMP9, AKT, p-AKT, p53, Cyclin D1, PCNA, β-actin, and secondary antibodies were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Cytotoxicity Assay, Flow Cytometry, Control, Over Expression

Journal: Translational Oncology

Article Title: Thermoradiotherapy reverses proliferation, metastasis, and anoikis resistance in glioblastoma through ITGA5-PI3K/AKT axis

doi: 10.1016/j.tranon.2025.102552

Figure Lengend Snippet: ITGA5 activates PI3K/AKT signaling by interacting with PI3K. ( A)Protein-protein interactions of ITGA5 and relevant factors (STRING). (B, C) IP and WB analyses show the interaction of ITGA5 and PI3K in GBM cells. (D, E) Protein levels of total and phosphorylated PI3K and AKT contents in ITGA5-OE and ITGA5-Ctrl GBM cells and tissues. β-actin served as the loading control. Protein levels of total and phosphorylated PI3K and AKT contents in sh-RNA, sh-ITGA5 and sh-ITGA5+ITGA5 cells (F) or sh-RNA and sh-ITGA5 tissues (G). β-actin served as the loading control. (H) Thermoradiotherapy reverses anoikis resistance in GBM by targeting ITGA5 via inhibiting the phosphorylation of PI3K/AKT. ITGA5-Ctrl: control groups for ITGA5-overexpression; ITGA5-OE: ITGA5-overexpression; sh-RNA: control groups for ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down; sh-ITGA5: ITGA5-knocking-down GBM cells complemented 1 μg ITGA5.

Article Snippet: Primary antibodies, PI3K, pPI3K, N-cadherin, Vimentin, E-cadherin, MMP2, Bax, and Bcl-2 were obtained from Cell Signalling Technology (Danvers, MA, USA), whereas ITGA5, MMP9, AKT, p-AKT, p53, Cyclin D1, PCNA, β-actin, and secondary antibodies were purchased from Proteintech (Wuhan, Hubei, China).

Techniques: Protein-Protein interactions, Control, Phospho-proteomics, Over Expression