Analyses of Intravesicular Exosomal Proteins Using a Nano-Plasmonic System

Transcription

1 Supporting Information Analyses of Intravesicular Exosomal Proteins Using a Nano-Plasmonic System Jongmin Park 1, Hyungsoon Im 1.2, Seonki Hong 1, Cesar M. Castro 1,3, Ralph Weissleder 1,4, Hakho Lee 1,2 * 1 Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA Department of Radiology, Massachusetts General Hospital, Boston, MA Massachusetts General Hospital Cancer Center, Boston, MA Department of Systems Biology, Harvard Medical School, Boston, MA Number of pages: 11 Number of figures: 10 S1

2 FDTD simulation for substrate engineering Fig. S1. SPR substrate engineering. Increasing substrate s refractive index moves the Au-substrate resonance towards longer wavelength. This makes it easier to track the Au-water peak. S2

3 Chip configuration Fig. S2. Nanoplasmonic chip configuration. Photographs of (a) Front and (b) backside of chip showing sensing arrays in a chip. The chip size is 2.5 cm 2.5 cm. The size of sensing arrays is 100 µm 100 µm and the inter-array distance is 2 mm. (c) The layout of the sensing array for processing two samples in a nanoplasmonic chip. S3

4 Chip fabrication Fig. S3. New fabrication method for scale-up production of inps chips. [STEP 1] A photoresist (PR) is spin- coated on a Si/SiN wafer, and the nanoholes are patterned via interference lithography. [STEP 2] The patterned wafer is dry-etched to make nanoholes in the SiN layer. [STEP 3] The Si-side of the wafer is lithographically patterned and wet-etched to define individual inps measurement sites. [STEP 4] Metal (Au/Ti) layers are directly deposited on the SiN side without further lithography steps. S4

5 Chip-to-chip variation of inps Fig. S4. Resonance peak positions from different inps chips. The coefficient of variation (CV) was <1% for all chips. S5

6 Detection limit of inps Fig. S5. Sensitivity of inps. Titration curve was drawn with various concentration of EVs. The detection limit was 10 4 EV. S6

7 Comparison of inps and ELISA Fig. S6. Comparison of inps and ELISA measurements. The correlation (R 2 ) between these assay is S7

8 Comparison of ovarian cells and their EVs protein profiles Fig. S7. Protein profiling of ovarian cancer cells and their secreting EVs. Transmembrane (CD63, EpCAM, EGFR) and intravesicular (AKT1, HSP90, HSP70, TSG101) protein levels were measured. Cellular protein expression of three ovarian cancer cell lines (OV420, CaOV3, and OVCAR3) and one normal cell lines (TIOSE6) were measured by flow cytometry. EVs secreted from those cells were analyzed by inps. Cellular and EV protein profiles show high correlation (R 2 = ). MFI, mean fluorescence intensity. S8

9 Drug treatment in ovarian cancer cell lines The concentrations for compound treatment were determined based on AKT and pegfr levels in OV90 and OV429 cell lines (Fig. S3 and S4) cells were seeded in 6 well plate 24 h before compounds treatment. OV90 and OV429 cells were treated by HSP90 inhibitor, 17-AAG (Selleck Chem) or EGFR inhibitor, Gefitinib (Selleck Chem) in varying concentrations, respectively, for 24 h and 48 h. Cells were washed with PBS and kept at -80 until use. 100 µl RIPA buffer with halt TM phosphatase inhibitor (Thermo) was added to each well for cell lysis and scrapped for cell lysate harvest followed by centrifugation at 14,000g, 4 for 5 mins. Supernatant was transferred to a new tube and the protein concentration was measured with BCA kit (Thermo). Cell lysate was mixed with 4x NuPAGE LDS sample buffer (Life Technologies) and boiled at 90 for 5 mins. The protein was dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred onto polyvinylidene fluoride membranes (PVDF, Invitrogen) and immunoblotted with antibodies against GAPDH (2118, Cell Signaling), HSP90 (k3720a, Biolegend), HSP70(4876, Cell signaling), AKT1 (Y89, Abcam), EGFR (ab24293, abcam), and p-egfr (ab5652, abcam). The concentration and incubation time of compounds were determined based on western blot data (17-AAG : 10 μm, 48 h ; Gefitinib : 20 μm, 48 h). Figure S8. Treatment of HSP90 inhibitor, 17-AAG. HSP90, HSP70 and AKT1 showed drastic changes at 10 μm for 48 h treatment in OV90 cell line. AKT1 level was fully decreased at 20 μm for 48 h treatment. S9

10 Figure S9. Treatment of EGFR inhibitor, Gefitinib. EGFR and p-egfr showed drastic changes at 20 μm for 48 h treatment in OV429 cell line. EGF (ab9697, abcam), a ligand protein of EGFR did not affected EGFR phosphorylation level in OV429 cell line. S10

11 Protein level in EVs upon drug treatment. OV90 and OV429 cells were seeded in 150Φ dishes 24 h before compounds treatment. OV90 and OV429 cells were treated by 10 μm HSP90 inhibitor, 17-AAG (Selleck Chem) or 20 μm EGFR inhibitor, Gefitinib (Selleck Chem) in vesicle-depleted medium (with 5% depleted FBS), respectively, for 48 h. Conditioned medium from 10 7 cells was collected and centrifuged at 300 g for 5 min. Supernatant was filtered through a 0.2 µm membrane filter (Millipore) and concentrated by 100,000 g for 1 h. After the supernatant was removed, the EV pellet was washed with PBS and centrifuged at 100,000 g for 1 h. The EV pellet was resuspended in PBS. 100ul RIPA buffer with halt TM phosphatase inhibitor (Thermo) was added to EV ( /ml) solution for lysis. EV lysates were incubated at 4 for 15 mins and followed by centrifugation at 14,000g, 4 for 5 mins. Supernatant was transferred to a new tube and the protein concentration was measured with BCA kit (Thermo). EV lysates were mixed with 4x NuPAGE LDS sample buffer (Life Technologies) and boiled at 90 for 5 mins. The protein was dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), transferred onto polyvinylidene fluoride membranes (PVDF, Invitrogen) and immunoblotted with antibodies against HSP90 (k3720a, Biolegend), HSP70(4876, Cell signaling), AKT1 (Y89, Abcam), EGFR (ab24293, Abcam), CD63 (556019, BD science), EpCAM (ab187270, Abcam), TSG101 (GTX118736, Genetex) and GAPDH (2118S, Cell signaling). Figure S10. CD63 and GAPDH level in EVs upon drug treatment. (a) 10 μm HSP90 inhibitor, 17-AAG, was treated OV90 cells for 48 h in conditioned media. EVs ( /ml) secreted from OV90 were collected and its lysates were analyzed by Western blot analysis. (b) 20 μm EGFR inhibitor, Gefitinib, was treated OV429 cells for 48 h in conditioned media. EVs ( /ml) secreted from OV429 were collected and its lysates were analyzed by Western blot analysis. S11