Mycoalert detection assay

We used the human hepatoblastoma
HepG2 cell line (American Type Culture Collection, ATCC, Manassas,
VA, USA) and human hepatocellular carcinoma cell line Alexander (PLC/PRF/5,
ATCC, Manassas, VA, USA) in this study. Cell cultures were cultivated
in Minimum Essential Medium Eagle (BioConcept Ltd., Switzerland) supplemented
with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, US), 1% l-glutamine 100× (200 mM stock, Serana Europe GmbH, Germany),
and 1% penicillin/streptomycin (Thermo Fisher Scientific, US). Cell
cultures were kept in a humidified 5% CO₂ atmosphere at
37 °C. The culture medium (EMEM) was changed once per week. Cells
were regularly checked for common culture contamination such as mycoplasma
using the MycoAlert Detection Assay (Lonza, Switzerland). All cell
lines were authenticated by short tandem repeat (STR) DNA profiling
(ATCC, Manassas, VA, USA).
Approximately 10⁵ HepG2
and Alexander cells in a volume of 30 μL were seeded into porous
collagen scaffolds. CSs were placed in a 12-well plate. After 1 h,
the fresh medium was added to cover the whole surface of CSs. For
all utilized assays in the study, cells were cultivated in CSs for
7 days in a humidified 5% CO₂ incubator at 37 °C,
and the culture medium was changed every other day. Cells seeded on
a standard glass-bottom dish (Cellvis, Mountain View, CA, USA) with
a 35 mm diameter served as the monolayer culture (MC) model.

The human PCa cell lines LNCaP and PC3 were obtained from the American Type Culture Collection. C4-2 cells were kindly provided by Prof. Thalmann (University of Berne, Switzerland) [24 (link)]. LAPC4 cells were provided by Dr A. Cato (University of Karlsruhe, Karlsruhe, Germany). LNCaP and C4-2 have been chosen as they represent a connected cell model system representing the castration sensitive (LNCaP) and castration-resistant (C4-2) PCa. Moreover, C4-2 express ARv7. LAPC4 has been selected as it expresses a wild type AR. PC3 has been chosen as AR and PSMA negative control. LAPC4, LNCaP, C4-2, and PC3 were cultured as described in Erb et al. 2020 [30 (link)]. Characteristics of the cell lines have been previously described in Table 1. All cell lines were maintained at 37 °C in 5% CO₂. Mycoplasma testing was performed using the Mycoalert Detection Assay (Lonza, Basel, Switzerland). STR profiling was used to verify cell line authentication.

Mouse Embryonic Fibroblasts (MEFs) were cultured in DMEM supplemented with 10% (v/v) fetal calf serum (FCS), 100 μM asparagine and 50 μM of β-mercaptoethanol (β-Met). HELA cells were cultured in DMEM media supplemented with 10% (v/v) FCS. T47D, MCF7, MDA-MB-231, NCI-H522, HOP62, HOP92, IGROV1, RXF393, 786-0, TK10, HCT116, PC3, MALME3M, LOXIMVI, KMS-28-BM, RPMI8226, KMS-12-PE, MOLT4, RS4;11, and SR cells were cultured in RPMI1640 supplemented with 10% (v/v) FCS. All cells were cultured humidified incubators maintained at 37 °C and 10% CO₂ for DMEM media or 5% CO₂ for RPMI media.
All mouse fibroblast cell lines were generated from E13.5 embryos derived from mice on an inbred C57BL/6 background, and transformed with SV40 large T antigen. BAX^−/−BAK^−/− HELA cells were generated from HELA CCL2 and deletion of BAX and BAK was confirmed by sequencing. All other cell lines were obtained from ATCC, DSMZ, or JCRB cell line repositories. All cell lines were confirmed to be mycoplasma negative using the MycoAlert detection assay (Lonza).
Where indicated, cells were cultured with the following compounds: doxycycline (Sigma), nocodazole (Sigma), cycloheximide (Sigma), QVD-OPh (MP Biomedicals), MG132 (Sigma).

Human metastatic Triple Negative Breast Cancer Cell Lines (TNBC-CL) MDA-MB-231 (CL 1) and MDA-MB-436 (CL 2) as well as a non-malignant cell line, HaCaT (immortalized human keratinocytes), were obtained from ATCC including the authentication certificate (genomic profiling) and were cultured in Dulbecco’s modified Eagle’s medium (Gibco Fisher Scientific). All cell lines were tested for Mycoplasma using Lonza’s MycoAlert detection assay. For TEX production, 4 × 10⁶ cells were cultured in 25 mL medium in a 150 cm² flask for 72 h. The supernatant was collected for sEV isolation as previously described^{37 (link)}. Pre-clarified supernatant was concentrated to 1 mL using a Vivaspin-20, 100,000 MWCO (Sartorius Corp, Bohemia, NY, USA) and placed on a Sepharose 2B column for sEV isolation by size exclusion chromatography (SEC)^{38 (link)}. The Jurkat cell line expressing surface CD8 protein was obtained from Dr H. Rabinowich (Department of Pathology, University of Pittsburgh, PA) and cultured in RPMI medium^{39 (link)}. All culture media were supplemented with 10% (v/v) exosome-depleted and heat-inactivated fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C and in an atmosphere of 5% CO₂ in air.

The human PCa cell lines LNCaP and PC3 were obtained from the American Type Culture Collection (ATCC). Prof. Thalmann (University of Berne, Berne, Switzerland) kindly provided the C4-2 cell line [23 (link)]. LNCaP and its subcell line C4-2 represent a castration sensitive (LNCaP) and castration-resistant (C4-2) phenotype. LNCaP, C4-2, and PC3 were cultured as described in Erb et al., 2020 [26 (link)]. The main characteristics of the used cell lines are listed in Table 1. Mycoplasma testing was performed using the Mycoalert Detection Assay (Lonza, Basel, Switzerland). STR profiling was used to verify cell line authentication. For the experiments, steroid hormone and growth factors withdrawal were achieved by a medium change to 5% dextran-coated charcoal treated FBS (FBSdcc; Thermo Fischer Scientific, Waltham, MA, USA). Androgen treatment was performed using the synthetic androgen R1881 (Sigma-Aldrich, St. Louis, MO, USA, R0908-10MG, Lot No: 085M4610V) dissolved in DMSO (20 µM stock solution), and aliquots were stored at −80 °C.

HeLa cells were obtained from the American Type Culture Collection (ATCC CCL-2). Cells were cultured in Minimum Essential Medium Eagle (BioConcept Ltd., Switzerland) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, US), 1% l-Glutamine 100 ×, 200 mM (Serana Europe GmbH, Germany) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific, US). Cell cultures were cultivated in a humidified 5% CO₂ atmosphere at 37 °C. Cell culture medium was replaced once a week. Cells were regularly checked for common culture contamination, such as Mycoplasma using MycoAlert Detection Assay (Lonza, Switzerland). HeLa cell line was authenticated by short tandem repeat (STR) DNA profiling (ATCC, Manassas, VA, USA).