Assembly of STV-oligonucleotide complexes

STV was combined with biotinylated oligonucleotides that included proteinTag sequences. Aliquots of recombinant STV (ThermoFisher Scientific) at a concentration of 1 μM were mixed with an equal volume of PBS containing either each one or a combination of all four biotinylated oligonucleotides (Supplementary Table 1) at a total concentration of either 100 nM or 10 μM and incubated at 4 °C overnight. The four separate STV-oligonucleotides complexes were combined into one, and both this pool and the preparation with all four oligonucleotides were diluted to a STV concentration of 1 nM in PBS.

Preparation of exosomes from cell culture supernatants or from human body fluids

Gastric cancer cell lines KATOIII (ATCC® HTB-103), AGS (ATCC® CRL-1739), MMK7(RIKEN) were cultured at 37 °C under 5% CO2 in RPMI1640 medium with 10% complete fetal bovine serum (FBS), penicillin-streptomycin, and glutamine. Before exosome isolation, cells were grown in RPMI1640 with 10% exosome-depleted FBS (EXO-FBS, System Biosciences) for 3–4 days up to 95% confluency. Cell culture media were collected and protease inhibitor (Complete Mini, Roche) was added to prevent degradation. Culture media were centrifuged at 3,000 × g for 10 min, and the supernatant was then centrifuged at 10,000 × g for another 10 min at 4 °C to remove cell debris. The supernatant was passed through a 0.45 µm filter and then ultracentrifuged at 100,000 × g for 2 h at 4 °C using a Beckman L8–70M ultracentrifuge. The pellet was resuspended and washed with cold filtered PBS and ultracentrifuged for 2 h again. The final exosome pellet was resuspended in 50–70 µl of PBS containing protease inhibitor and stored at −80 °C until use. The concentration of total protein in isolated exosomes was measured using a Pierce BCA protein assay kit (Thermo Scientific) and a dot-it-spot-it kit (Maplestone AB, Sweden). Lyophilized exosomes from BLCL (EBV transformed lymphoblastoid B cell line, HBM-BLCL-30/2), K562 (pleural effusion, leukemia chromic myelogenous, HBM-K562–30/2), DAUD1 (human burkitt lymphoma, HBM-DAUDI-30/2), U87MG (human glioblastoma, HBM-U87–30/2), SKNSH (human neuroblastoma, HBM-SK-30/2), HEK293 (human embryonic kidney, HBM-HEK293–30/2), HCT116 (human colon carcinoma, HBM-HCT-30/2), COLO1(human colon carcinoma, HBM-COLO-30/2), A549 (lung carcinoma, HBM-A549–30/2), PC3 (human prostate adenocarcinoma grade IV, HBM-PC3–30/2), BPH-1 (human prostatic hyperplasia), MM1 (human melanoma, HBM-BPH-30/2), and from serum of healthy donors (HBM-PES-100) were all purchased from HansaBioMed Life-sciences LLC.

To purify prostasomes, human seminal plasma was centrifuged at 3,000 × g for 10 min, followed by 10,000 × g for 30 min at 4 °C to pellet cell debris. The supernatant was ultracentrifuged at 100,000 × g for 2 h at 4 °C and the pellet, containing exosomes, was resuspended in PBS. The resuspended pellet was further purified by size-exclusion chromatography on a Superdex 200 gel-filled XK16/70 column (GE Healthcare), followed by density gradient separation (prostasomes were recovered in the density range 1.13–1.19 g/ml). The prostasome concentration was adjusted to 2 mg/ml measured by Pierce BCA protein assay kit and were kept at −70 °C until use.

Antibodies and oligonucleotides

The antibody preparation directed against streptavidin was purchased from Thermo Scientific (S10D4). The sources of antibodies directed against surface proteins on exosomes are summarized in Supplementary Table 2. The oligonucleotides used in this study were purchased from IDT or Solulink (Supplementary Table 1).

Preparation of antibody-oligonucleotide conjugates (PBA probes)

The conjugation of oligonucleotides to antibodies (Supplementary table 2) was performed as follows: Twenty μg of each antibody was activated by adding 1 μl of 4 mM Sulfo-SMCC (Thermo Scientific) in dimethyl sulfoxide (DMSO; Sigma–Aldrich), and incubating at room temperature (RT) for 2 h. After 1 h of antibody activation, 3 μl of each 5’ thiol-modified oligonucleotide at a concentration of 100 μM was reduced by adding 12 μl of 100 mM DTT (Sigma–Aldrich) in 1x PBS with 5 mM EDTA, and incubating at 37 °C for 1 h. The activated antibodies and reduced oligonucleotides were separately purified using Zeba Spin Desalting Plates, 7 K MWCO (Thermo Scientific) according to the manufacturer’s recommended procedure. Each purified antibody was then mixed with one type of oligonucleotide (Supplementary Table 1), and directly followed by dialysis in a Slide-A-Lyzer MINI Dialysis Device, 7 K MWCO, 0.1 ml (Thermo Scientific) against 5 l PBS with constant stirring by a magnetic bar at 4 °C overnight. The conjugates were stored at 1 μM antibody concentration in PBS with 0.1% BSA at 4 °C.

Preparation of RCA products

Padlock oligonucleotides, including a 15 nt random sequence (100 nM) were ligated into circular DNA strands in the presence of template (100 nM) in 1x phi29 buffer ((Thermo Scientific), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween 20, 1 mM DTT) containing 0.01 U/μl T4 ligase (Thermo Scientific), and 1 mM ATP (Thermo Scientific). The ligation was performed at 37 °C for 30 min. To remove unligated padlock oligonucleotides, exonuclease I and exonuclease III (Thermo Scientific) were added to the ligation mix to a concentration of 0.2 U/μl and 2 U/μl, respectively. The reactions were incubated at 37 °C for 30 min and terminated by incubation at 85 °C for 20 min. Then the RCA primer (the same oligonucleotide used as ligation template) was adjusted to a concentration of 100 nM and incubated at 37 °C for 20 min to reanneal to the circular DNA strands. The RCA reactions were initiated by adding d(A, U, G, C)TP at concentrations of 1 mM and phi29 polymerase (Thermo Scientific) at 0.1 U/μl. RCA was performed at 37 °C for 10 min, and terminated by heating at 65 °C for 10 min. The RCA products were kept at −20 °C until use. The RCA products were characterized with Nanoparticle Tracking Analysis NTA (Malvern Nanosight NS300) according to the manufacturer’s instructions.

Capturing STV-biotin oligonucleotide complexes

For each reaction, 200 ng anti-STV antibodies, diluted in 50 μl 100 mM carbonate buffer (pH 9.6), was added to 8-well RoboStrip (847–0501000103, Analytik Jena) and incubated overnight at 4 °C. After two washes with 100 μl washing buffer (1x PBS with 0.05% Tween20 (Sigma–Aldrich)), 50 μl blocking buffer (1x PBS with 1% BSA (Sigma–Aldrich)) was added and the plates were incubated at 37 °C for 1 h. After removing the blocking buffer, pre-assembled STV-oligonucleotides complexes were diluted to 10 pM (according to the concentration of STV) in 20 μl washing buffer, and added to the antibody-immobilized microplates, and incubated for 1 h at RT.

Probing and capturing exosomes

For each reaction, 50 ng biotinylated CTB (C-34779, Thermo Scientific) was diluted in 25 μl PBS and incubated overnight in STV-coated PCR tubes (PCR0STF-SA5/100, Biomat). The wells in the plates were then washed twice with 100 μl washing buffer. Exosomes from each sample source or cell line was mixed with all antibody conjugates (20 nM for each), directed against surface proteins, in PBA buffer (PBS, 0.05% Tween 20, 5 mM EDTA, 1 mg/ml salmon sperm DNA (15632011, ThermoFisher Scientific) and 1% BSA) at 4 °C overnight. After incubation, 1 μl of the exosome-antibody conjugate complexes were diluted in 25 μl PBA buffer, and 20 μl was incubated in wells coated with biotinylated CTB for 15 min at RT. For replicate measurements, 2 μl of exosomes were diluted in 50 µl PBA buffer, and 20 μl were added into two separated wells.

Proximity barcoding

The captured STV-oligonucleotide complexes or exosomes with antibody conjugates were washed twice with 100 μl washing buffer. Then 25 μl of the RCA products at a concentration of 1 nM and 500 nM blocking oligonucleotide was added to the reaction wells and incubated at 37 °C for 15 min. The blocking oligonucleotides were used to prevent the DNA polymerase from extending to adjacent copies of monomers in the RCA products containing the complexTags. After two washes with 100 μl washing buffer, 25 μl of T4 DNA polymerase buffer containing 1.25 unit of T4 DNA polymerase and 100 μM d(A, T, G, C)TP was added and the reaction mix was incubated at RT for 15 min.

Amplification of complex-tagged extension products from the antibody-conjugated oligonucleotides

The reaction mixes were washed and 30 μl of PCR mix containing 1x Phusion HF Buffer (2 mM MgCl 2 , 0.2 mM d(A,T,G,C)TP, 1× SYBR Green I, 1% DMSO, 500 nM PCR primers (pba-fwd and pba-rev), 0.02 U/μl Phusion polymerase and 0.02 U/μl Uracil-DNA glycosylase (all from Thermo Scientific) was added to each reaction well. The plate was then transferred to a thermal cycler (MX3005; Stratagene) for qPCR with an initial incubation of 37 °C for 15 min, then 95 °C for 2 min, followed by 30 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s.

Sample indexing and library preparation for sequencing

After PCR amplification of extension products from PBA reactions, 1 μl of the amplification reaction was spiked in 10 μl of index PCR mix containing 10 μM each of fwd-index and rev-index primer pairs, and the reactions were incubated for an initial 2 min at 95 °C, followed by two cycles of 95 °C for 15 s, 60 °C for 60 s, and 72 °C for 60 s. The indexed PCR products were diluted 20 times into new PCR mixes containing 1x Phusion HF Buffer (Thermo Scientific), 0.2 mM d(A, T, G, C)TP (Thermo Scientific), 1× SYBR Green I (Thermo Scientific), 1% DMSO, 500 nM PCR primers (library-fwd and library-rev) and programmed for an initial 2 min at 95 °C, and 15 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were pooled and purified using QIAquick PCR Purification Kit. The purified DNA was sequenced using MiSeq Reagent Kit v2, 300 cycles by MiSeq or NextSeq Reagent Kit 75SE, NextSeq (Illumina).

SP-PLA for prostasome detection in 10% human plasma

Biotinylated CD26 and CD59 were prepared using ChromaLink Biotin Labeling Reagent (Solulink B-1007–105) with 20-fold molar excess of biotin reagent over antibody according to the manufacturer’s protocol. Before biotinylation, antibodies were purified using Zeba Spin Desalting Column, 7 K MWCO (Thermo Scientific, 89882) according to the manufacturer’s recommended procedure and in PBS at concentration of 2 mg/ml. After biotinylation, the antibodies were purified again with Zeba Spin Desalting Column to remove excess biotin reagents. Dynabeads MyOne Streptavidin T1 (100 μl, 1 mg, ThermoFisher Scientific, 65601) was washed twice with 500 μl washing buffer (PBS with 0.05% Tween 20), and 200 μl of 50 nM CTB-biotin conjugates (C34779) were added to the beads and the suspensions were incubated for 1 h at RT on a rotator. The beads were then washed twice with 500 μl PBST and reconstituted in 200 μl PBS containing 0.1% BSA. PLA probes (CD26-SLC1 and CD59-SLC2) were prepared by mixing 100 nM of biotinylated CD26 and CD59 with 100 nM streptavidin-conjugated oligonucleotides (SLC1 and SLC2, Solulink) at same volume separately and incubated for 30 min at RT. Then two probes were diluted in PLA buffer (1 mM D-biotin, 0.1% purified BSA, 0.05% Tween 20, 100 nM goat IgG, 0.1 μg/μl salmon sperm DNA, 5 mM EDTA in PBS) to 1 nM, incubated for 30 min at RT and mixed at same volume. The final concentration for each probe is 500 pM.

Purified prostasomes were serially diluted in 10% human plasma (in PLA buffer). Each dilution series included a negative control with only 10% human plasma in PLA buffer. Diluted prostasomes (45 μl) were mixed with a suspension of CTB-coated beads (5 μl) and the reactions were incubated for 1.5 h at RT on an end-to-end rotator. The beads were then washed twice in 100 μl PBST using a 96-well magnetic rack. Fifty μl of 500 pM pairs of PLA probes were added into each reaction well, incubated for 1.5 h at RT, and the beads were washed twice in 100 μl PBST. A 50 μl ligation and PCR mixture (1 × PCR buffer, 2.5 mM MgCl 2 , 0.5 × Sybr Green I, 0.1 μM BioFwd primer, 0.1 μM BioRev primer, 0.1 μM BioSplint, 0.08 mM ATP, 0.2 mM dNTP (with dUTP), 0.03 U/μl AccuStart Taq DNA polymerase, 0.01 U/μl T4 ligase and 0.002 U/μl Uracil-DNA glycosylase) was added to each well. Real-time PCR was performed in MX3005 cycler (Life technologies, CA, USA) programmed at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.

Data analysis

The BCL files for each sample were converted to fastq formats by using bcl2fastq (Illumina) with pair indexes. Then the complexTag, proteinTag, and moleculeTag were extracted from three fixed segments within each read. Reads with only one count were removed from the analysis. The tags were then sequentially sorted according to complexTag, proteinTag, and moleculeTag using an in-house developed Perl script. The number of exosomes with given combinations of proteins were calculated using an in-house developed R script. The t-SNE algorithm applied was implemented in the R package Rtsne, and all the parameters were set to default in our pipeline.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.