Extra-domain B of fibronectin as an alternative target for drug delivery and a cancer diagnostic and prognostic biomarker for malignant glioma

Extra-domain B of fibronectin (EDB-FN) is an alternatively spliced form of fibronectin with high expression in the extracellular matrix of neovascularized tissues and malignant cancer cells. In this study, we evaluated the practicality of using EDB-FN as a biomarker and therapeutic target for malignant gliomas (MGs), representative intractable diseases involving brain tumors. Methods: The microarray- and sequence-based patient transcriptomic database 'Oncopression' and tissue microarray of MG patient tissue samples were analyzed. EDB-FN data were extracted and evaluated from 23,344 patient samples of 17 types of cancer to assess its effectiveness and selectivity as a molecular target. To strengthen the results of the patient data analysis, the utility of EDB-FN as a molecular marker and target for MG was verified using active EDB-FN-targeting ultrasmall lipidic micellar nanoparticles (~12 nm), which had a high drug-loading capacity and were efficiently internalized by MG cells in vitro and in vivo. Results: Brain tumors had a 1.42-fold cancer-to-normal ratio (p < 0.0001), the second highest among 17 cancers after head and neck cancer. Patient tissue microarray analysis showed that the EDB-FN high-expression group had a 5.5-fold higher risk of progression than the EDB-FN low-expression group (p < 0.03). By labeling docetaxel-containing ultrasmall micelles with a bipodal aptide targeting EDB-FN (termed APTEDB-DSPE-DTX), we generated micelles that could specifically bind to MG cells, leading to superior antitumor efficacy of EDB-FN-targeting nanoparticles compared to nontargeting controls. Conclusions: Taken together, these results show that EDB-FN can be an effective drug delivery target and biomarker for MG.


Competition assay
EDB-FN high expression cells (U87MG and U251MG) and EDB-FN low expression cells (MCF7 and B16F1) were grown on glass coverslips until reaching ~80% confluence.
Cells were then pre-treated with different concentrations (100 μg/mL and 500 μg/mL) of free APTEDB peptide for 30 min. Then, rhodamine B-labeled APTEDB-DSPE was added and cells were incubated for an additional 30 min. Thereafter, cells were washed with PBS, fixed with 4% (w/v) paraformaldehyde, and mounted on microscope slides for viewing under a confocal microscope.

Transfection of small interfering RNA (siRNA)
Specific siRNAs for EDB-FN and the scrambled control siRNA were purchased from Bioneer (Daejeon, Republic of Korea). Target sequences of EDB-FN siRNAs used in RNA interference were as follows: sense; ACAGUCCCAGAUCAUGGAG, antisense; CUCCAUGAUCUGGGACUGU. For transfection experiments with Lipofectamine 2000 (Invitrogen, CA, USA), cells were seeded into 96-well or 6-well plates at 60-70% confluence after overnight growth. Lipofectamine-siRNA complexes were prepared according to the manufacturer's instructions. Transfection efficiency was analyzed 48 h later.

In vitro cellular uptake of the APTEDB-DSPE micellar nano-DDS
To confirm intracellular uptake dependent on the amount of EDB-FN expression, U87MG cells were transfected with control siRNA or EDB-FN siRNA as described above.

In vivo uptake and toxicity of APTEDB-DSPE.
To evaluate the tissue uptake of the APTEDB-DPSE micellar nano-DDS in vivo, U87MG cells were injected into the right flank of BALB/c nude mice (n = 3 mice per group) at 5 × 10 6 cells/mouse. After 3 weeks, tumor growth was measured, and the tumor volumes were determined to be 80-120 mm 3 . Then, 200 µg of the PEG2000-DSPE micellar nano-DDS or APTEDB-DSPE micellar nano-DDS was injected into each mouse, and at predetermined time points (6, 12, 24, and 48 h), the tumor uptake of rhodamine B-labeled micelles was compared using an IVIS in vivo imaging system (PerkinElmer, MA, USA).
To verify the safety of APTEDB-DSPE, mouse weight was confirmed before and after the experiment. After the end of the experiment, we euthanized the mice to collect all major organs (heart, liver, spleen, lung, and kidney) for H&E staining.

Immunohistochemistry for frozen samples of orthotopic xenograft model
The brain slices of orthotopic xenograft model attached to the slide glass were washed twice with cold PBS, and then blocked and permeabilized in blocking buffer (PBS containing 0.3% Triton X-100 and 2% BSA) for 1 h. The primary antibody specific for EDB-FN (ab154210; Abcam, MA, USA) was diluted 1:100 in blocking buffer and incubated overnight at 4 °C with the tissue. After washing with PBS, it was incubated for 1 hour at room temperature with Alexa Fluor 488 conjugated secondary antibody (A11001; Invitrogen, CA, USA) diluted 1:200 in blocking buffer. The tissue was counterstained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, NY, USA), covered with a cover slide, and analyzed using a confocal laser scanning microscope and slide scanner (Axio Scan.Z1; Carl Zeiss, NY, USA). Table   Table S1. EDB-FN expression-related prognostic differences among GBM patients