Journal: International Journal of Medical Sciences

Article Title: Targeted inhibition of integrin αVβ3 induces cytotoxicity and suppresses migration ability in ovarian cancer cells and tumor spheroids

doi: 10.7150/ijms.103141

Figure Lengend Snippet: Effect of CGT and knockdown of integrin αVβ3 on cell viability in ovarian cancer cells. (A) SKOV3 and TOV-21G (4 × 10 3 cells/well of 96-well plate) were treated with various concentrations of CGT (0, 5, 10, and 20 μM) for 48h. Cell viability was analyzed by MTT assay. (B) The indicated cells were harvested by trypsin, stained with trypan blue, and counted under an inverted microscope at indicated times. (C) After combined treatment with CGT and cisplatin for 48h, MTT assay was performed to analyze the cell viability of SKOV3 cells. (D) Combination index of cotreatment of CGT and cisplatin for 48 on SKOV3 cells was calculated by software Compusyn 1.0. The definitions of combination index are synergistic effect (combination index < 0.9), additive effect (0.9 < combination index < 1.1), and antagonism (combination index > 1). (E) Representative images of wound healing of SKOV3 cells with or without CGT treatment. (F) Values of percentage wound closure. The areas lacking cells were analyzed by ImageJ. The symbol '*' indicates P < 0.05.

Article Snippet: The FITC conjugated anti-integrin αVβ3 antibody (MAB1976F, Merck, Darmstadt, Germany) was used to detect the integrin αVβ3 in cells.

Techniques: Knockdown, MTT Assay, Staining, Inverted Microscopy, Software

Journal: International Journal of Medical Sciences

Article Title: Targeted inhibition of integrin αVβ3 induces cytotoxicity and suppresses migration ability in ovarian cancer cells and tumor spheroids

doi: 10.7150/ijms.103141

Figure Lengend Snippet: Expression and localization of integrin αVβ3 in ovarian cancer cells. (A) Total cell lysates of SKOV3 and TOV-21G cells (4 × 10 5 cells of 60 mm dish) were used to investigate the indicated protein expression Western blot assay. (B) SKOV3 and TOV-21G cells were stained with FITC-conjugated integrin αVβ3 antibody and DAPI, and investigate the distribution of integrin αVβ3 in both ovarian cancer cells. (C) Fluorescence intensity of nuclear or whole cell was analyzed by ImageJ. (D) The cells were stained with FITC-conjugated anti-integrin αVβ3 antibody. Non-fluorescent anti-integrin αVβ3 antibody was performed as competing antibody. The unstained and stained cells were analyzed by flow cytometry. The symbol '*' indicates P < 0.05. N.S., not significant.

Article Snippet: The FITC conjugated anti-integrin αVβ3 antibody (MAB1976F, Merck, Darmstadt, Germany) was used to detect the integrin αVβ3 in cells.

Techniques: Expressing, Western Blot, Staining, Fluorescence, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: Targeted inhibition of integrin αVβ3 induces cytotoxicity and suppresses migration ability in ovarian cancer cells and tumor spheroids

doi: 10.7150/ijms.103141

Figure Lengend Snippet: Effect of CGT and knockdown of integrin αVβ3 on expressions of downstream protein. (A) The sample of Figure A and Figure C are the same. After treatment of various concentrations of CGT (0, 5, 10, and 20 μM) for 48h, total cell lysates of SKOV3 cells (4 × 10 5 cells of 60 mm dish) were analyzed by Western blot assay to detect the protein expressions of integrin αV and β3. (B) The protein expressions of phospho-FAK, CD44, and PD-L1 in SKOV3 cells after treating with CGT for 48h were analyzed by Western blot assay. (C) Western blot assay was performed to detect the expressions of the indicated protein in SKOV3 shLuc, shITGAV, shITGB3, and shITGAVB3 cells. β-actin served as a loading control. The results showed correlations between the gene expression of integrin αV and (D) CD274 (PD-L1) or (E) CD44 by the OncoDB database.

Article Snippet: The FITC conjugated anti-integrin αVβ3 antibody (MAB1976F, Merck, Darmstadt, Germany) was used to detect the integrin αVβ3 in cells.

Techniques: Knockdown, Western Blot, Control, Gene Expression

Journal: International Journal of Medical Sciences

Article Title: Targeted inhibition of integrin αVβ3 induces cytotoxicity and suppresses migration ability in ovarian cancer cells and tumor spheroids

doi: 10.7150/ijms.103141

Figure Lengend Snippet: Effect of CGT on apoptosis induction. (A) After treatment with CGT (0, 5, and 20 μM) for 48 h, SKOV3 cells (4 × 10 5 cells of 60 mm dish) were stained with annexin V-FITC/PI and analyzed by flow cytometry. (B) Mitochondrial membrane potential in SKOV3 cells (4 × 10 5 cells of 60 mm dish) treated with CGT (0, 5, and 20 μM) for 48 h was analyzed by fluorescence microscope and flow cytometry. (C) The sample of Figure A and Figure C are the same. The protein expressions of cleaved PARP and cleaved caspase-7 in SKOV3 cells after treating with CGT for 48h were analyzed by Western blot assay. β-actin served as a loading control.

Article Snippet: The FITC conjugated anti-integrin αVβ3 antibody (MAB1976F, Merck, Darmstadt, Germany) was used to detect the integrin αVβ3 in cells.

Techniques: Staining, Flow Cytometry, Membrane, Fluorescence, Microscopy, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Targeted inhibition of integrin αVβ3 induces cytotoxicity and suppresses migration ability in ovarian cancer cells and tumor spheroids

doi: 10.7150/ijms.103141

Figure Lengend Snippet: Effect of CGT on cell migration of tumor spheroid. (A) The spheroids with or without CGT treatment were removed to the 6-well plate for attachment and migration. (B) The spreading area of SKOV3 cells migrated from the tumor spheroid was measured by ImageJ software. (C) After attachment for 72h, the tumor spheroid and migrated cells was fixed and stained by phalloidin FITC reagent for detect the F-actin. The symbol '*' indicates P < 0.05.

Article Snippet: The FITC conjugated anti-integrin αVβ3 antibody (MAB1976F, Merck, Darmstadt, Germany) was used to detect the integrin αVβ3 in cells.

Techniques: Migration, Software, Staining

Journal: Cancer Research Communications

Article Title: Targeting Pancreatic Cancer Cell Stemness by Blocking Fibronectin-Binding Integrins on Cancer-Associated Fibroblasts

doi: 10.1158/2767-9764.CRC-24-0491

Figure Lengend Snippet: BsAb improves the targeting of FN-binding integrins on CAFs. A, BsAb for dual recognition of αvβ3 and α5β1 heterodimers. Schematic depicts the design of a novel BsAb with monovalent recognition of two antigens, integrins αvβ3 and α5β1. The Fab domains for the BsAb are identical to the “control” bivalent (i.e., monospecific) mAb recognizing αvβ3 (derived from etaracizumab) and α5β1 (derived from volociximab). B, BsAb binding to cells compared with commercial and control antibodies. Flow cytometry plots show the binding of each antibody to CAF-1299 and CAF-1424. C, BsAb shows improved binding to CAFs compared with constituent mAbs. The graph shows median fluorescence intensity (MFI) for antibody binding to CAF-1299 and CAF-1424 cells. Bars and error bars represent SD from n = 3 independent experiments.

Article Snippet: Anti-integrin αvβ3 antibody (LM609; 10 μg/mL for flow cytometry) was produced in the Cheresh Lab and is also commercially available (Millipore, MAB1976, RRID: AB_2296419).

Techniques: Binding Assay, Control, Derivative Assay, Flow Cytometry, Fluorescence

Journal: Cancer Research Communications

Article Title: Targeting Pancreatic Cancer Cell Stemness by Blocking Fibronectin-Binding Integrins on Cancer-Associated Fibroblasts

doi: 10.1158/2767-9764.CRC-24-0491

Figure Lengend Snippet: Dual blockade of integrins αvβ3/α5β1 can prevent and reverse CAF-ECM assembly. A, Function-blocking antibodies targeting FN receptors prevent CAF assembly of FN/COL fibers. CAF-1299 cells were incubated with control IgG vs. indicated antibodies for 72 hours and then processed for immunostaining to examine FN and COL. Images are representative of at least three independent experiments. The graph shows the quantification of staining as mean ± SD for each marker that was measured as % area for each experiment and then normalized to IgG control. *, P < 0.05 using one-sample t test. B, Integrin-targeted antibody can disrupt preexisting CAF-produced ECM. CAF-1299 were plated and allowed to produce ECM for 72 hours before adding control IgG vs. indicated antibodies for an additional 72 hours. Samples were then processed for immunostaining to examine FN and COL. The graph shows the mean ± SD staining for each marker measured as % area in each experiment and normalized to IgG control. *, P < 0.05 using the one-sample t test. C, BsAb prevents upregulation of connective tissue growth factor (CTGF), a protein that regulates cell proliferation, migration, and adhesion. CAF-1299 cells were plated and allowed to produce ECM for 72 hours and then treated with antibody for another 72 hours before cells were removed to leave behind a cell-free ECM atop which PANC1 cells were then plated. After 24 hours, the PANC1 cells were lysed and prepared for immunoblotting to detect the protein expression of CTGF. Blots are representative of at least three independent experiments. Tx, treatment.

Article Snippet: Anti-integrin αvβ3 antibody (LM609; 10 μg/mL for flow cytometry) was produced in the Cheresh Lab and is also commercially available (Millipore, MAB1976, RRID: AB_2296419).

Techniques: Blocking Assay, Incubation, Control, Immunostaining, Staining, Marker, Produced, Migration, Western Blot, Expressing

Journal: Cancer Research Communications

Article Title: Targeting Pancreatic Cancer Cell Stemness by Blocking Fibronectin-Binding Integrins on Cancer-Associated Fibroblasts

doi: 10.1158/2767-9764.CRC-24-0491

Figure Lengend Snippet: CAF-produced FN boosts tumor initiation (summary schematic). CAFs produce a dense and reactive stroma that supports tumor initiation and progression. This project establishes the link between cell surface integrins and FN as a lynchpin for the construction of protumor ECM that can account for the ability of CAFs to support tumor initiation for multiple in vitro and in vivo models. Knockdown or antibody blockade of integrins αvβ3 and/or α5β1 can disrupt FN fiber assembly. FN fibers are known to function as a scaffold that mediates the assembly of other ECM proteins and the incorporation of protumor-secreted factors. Knockdown of αvβ3/α5β1 or their dual blockade using a novel BsAb can disrupt this cascade by preventing the initial assembly of FN fibers. This approach has broad potential as a novel strategy to target the aberrant fibrosis that exacerbates the progression of cancer and fibrotic disease.

Article Snippet: Anti-integrin αvβ3 antibody (LM609; 10 μg/mL for flow cytometry) was produced in the Cheresh Lab and is also commercially available (Millipore, MAB1976, RRID: AB_2296419).

Techniques: Produced, In Vitro, In Vivo, Knockdown