Polybrene

In order to genetically label EVs, mEER and MOC2 cell lines were transduced as previously reported by our group [49 (link)]. Briefly, LV transfer plasmids were propagated in Escherichia coli DH5α. Maxiprep was performed with Endo-free Macherey-Nagel kit. Unconcentrated lentiviral vectors were generated. MOC2 and mEER cells were seeded at a concentration of 10⁵ cells per well in a 6-well plate and transduced with LV vector supernatants (1:1 ratio with complete media) in the presence of 1 μg/ml polybrene (Millipore). For the simultaneous in vivo detection of both tEVs^total and tEVs^CSC, mEER cell line was consecutively transduced with SS-mSca-LPETGG: mCMV-PGK:CD63-eGFP and ALDH1A1:SrtA LV vectors in the presence of 1 μg/ml polybrene (Millipore). Engineered cell lines were cultured under the same conditions than the ones described for the parental cell lines and propagated at 37°C and 5% CO₂ in a humidified incubator.

Self-inactivating replication of viral particles were produced by calcium phosphate-mediated transfection of packaging cells (HEK293T) with the appropriate amounts of transfer plasmid and HIV-1 lentiviral packaging constructs pDEL, pREV, and VSV-G. Supernatants were harvested 48 h after transfection, 0.22 μm filtered, concentrated by ultracentrifugation (50,000 × g; 2,20 h), and titrated by limiting dilution assay in HEK293T.
Human CD34⁺ cells were infected at a MOI (multiplicity of infection) of 2 with 8 µg/ml of polybrene (Millipore, Billerica, MA, USA) and cultured overnight in a 37 °C incubator at 8% CO₂. Virus-containing media was then removed and replaced with fresh one supplemented with cytokines (G-CSF and GM-CSF at 40 ng/ml each). The cells were then differentiated for 4 days. Human CD14⁺ cells were infected at a MOI of 1 with 8 µg/ml of polybrene (Millipore, Billerica, MA, USA) for 4 h by spin-inoculation (2000 rpm; 37 °C). Transduced cells received fresh media and were incubated overnight in a 37 °C incubator at 5% CO₂.

We performed lentiviral short hairpin (shRNA) transduction of the NEPC patient-derived organoid (PDO) WMC154 with lentivirus packaged with shRNA targeting CELSR3 (targeting sequence in the 3′ untranslated region) or scrambled sequence as a control as we have described previously (31 (link)). Briefly, NEPC PDO WMC154 were dissociated with TrypLE (Gibco) and resuspended in organoid medium (38 (link)) containing Polybrene (Millipore) and Y27632 (Selleckchem, S1049). The dissociated organoid cells were combined with viral suspension and centrifugated at 600 × g, 32°C for 60 minutes. The organoid/virus mix was then incubated at 37°C overnight. Organoid cells were subsequently collected, resuspended in 120 μL of Matrigel (Corning) and seeded in a 24-well plate. Antibiotic selection was performed using 1 μg/mL puromycin (Thermo Fisher Scientific) for 7 days. LNCaP-N-Myc cells were transduced in 6-well plates in medium containing Polybrene (Millipore). Cells were selected with 2 μg/mL puromycin (Thermo Fisher Scientific) for 7 days and grown without puromycin 1 for 6 weeks.

The shRNA sequences were designed according to the RNA Consortium’s recommendation (http://www.broadinstitute.org/rnai/trc) and cloned into the lentiviral vector pLKO.1-puro at the Age I and EcoR I digestion site. Each individual shRNA vector (500 ng) was co-transfected into 293T cells with the packaging plasmids pMDLg/pRRE (250 ng) and pRSV-Rev (250 ng) together with the envelope plasmid pMD2.G (250 ng) into 293T cells (200,000 cells) seeded into six-well plate 24 h before transfection, using the FuGENE 6 transfection reagent (Roche) and Opti-MEM, according to the manufacturer’s instruction. Medium was changed 24 h after transfection. Supernatants containing lentivirus was then collected after 48 and 72 h post transfection, combined, and filtered through a 0.45 mm nitrocellulose filter (Thermo). The Jurkat cells were infected with lentivirus in the presence of polybrene (8 μg/mL: Millipore) by centrifugation at 1300 rcf for 1.5 h. Neuroblastoma cells were transduced with lentivirus in the presence of polybrene (8 μg/mL: Millipore) for 2 h incubation followed by toping up fresh medium. Approximately 500,000 cells were infected with 1 mL of virus medium. The cells were then selected by the addition of puromycin (0.7 μg/mL for Jurkat cell line; 0.5 μg/mL for neuroblastoma cell lines) for at least 36 h after infection. shRNA sequences are shown in Supplementary Data 3.

The lentiviral vector was produced by transient co-transfection of HEK293T cells with a four-plasmid packaging system and concentrated using Lenti-X Concentrator (Clontech Laboratories, Mountain View, CA) as previously described^{29 (link)}.
For transduction of HeLa EGFP-654 cells, the cells were transduced with the lentiviral vectors at various multiplicity of infection (MOI) in the presence of 4 μg/mL polybrene (Hexadimethrine bromide; Sigma-Aldrich).
For transduction of CD34 + cells, the cells were transduced with lentivirus at an MOI of 150 in the presence of 8 μg/mL polybrene at day 3 of first phase. Transduced cells were selected by treated with 0.5 μg/mL puromycin (Clontech Laboratories) at 72 hr after transduction and continued for 2 days.

Human PCa PC3 and C4-2B cells were plated in 24-well plates (5×10⁴ cells/well) overnight. The luciferase reporter lentiviral system (GenePharma, Shanghai, China) were diluted in 0.4ml (10⁸ TU/ml) complete medium containing 5μg/ml polybrene (Sigma, St. Louis, MO) and added to the cells for incubation for 24 hours at 37°C, followed by incubation in freshly complete medium for another 48 hours. Cell sorting collected strong GFP positive cells by Flow Cytometry.
RM1 cells were infected with the luciferase reporter lentiviral particles we built above in the presence of 5 μg/ml polybrene for 48 hours and positive clones were selected by 4μg/ml Blasticidin (Sigma) for at least 2 weeks.
PC3-luc, C4-2B-luc and RM1-luc cell lysates were collected and the luciferase activity assay was performed with Luciferase Assay System (Promega). Cell lysates and substrate of luciferase were added together in a microplate, the luciferase activity was recorded by luminometer reader (Glomax, Promega) and protein concentration of cell lysis was detected by BCA assay.

For generation of knockdown cell lines, 293 T cells were seeded at a density of 1 × 10⁶ cells on 100 mm dishes. After 24 h, cells were transfected with pSUPER-CTRL shRNA or pSUPER-CCN3/GPNMB shRNAs along with pCMV-VSVG and pCMV-Gag-Pol (8:3:4 mass ratio). After 48 h, retroviral particles were collected from the culture medium of 293 T cells. Hs578T, MDA-MB-231, MDA-MB-436 and MDA-MB-231 LM1 cells were treated with media containing retroviral particles and 10 μg/mL of polybrene (Sigma, St. Louis, MO, USA). CTRL and CCN3/GPNMB knockdown clones were selected by treating cells with puromycin. The selected clones were subjected to western blot and RT-qPCR to confirm their decreased expression of target genes. For generation of overexpression cell lines, 293 T cells were seeded at a density of 1 × 10⁶ cells on 100 mm dishes. After 24 h, cells were transfected with pBMN-EGFP-vec or pBMN-EGFP-CCN3 along with pCMV-VSVG and pCMV-Gag-Pol (8:3:4 mass ratio). After 48 h, retroviral particles were collected from the culture medium of 293 T cells. HCC1806 cells were treated with media containing retroviral particles and 10 μg/mL of polybrene (Sigma, St. Louis, MO, USA). Cell lines were constructed by sorting only the cells expressing EGFP using flow cytometry.