TEVs were obtained from supernatant derived from the culture of the colon cancer cell lines SW1116 and HCT116 (American Type Culture Collection). Briefly, cells were cultured at 37°C, without CO₂ (SW1116) or with 5% CO₂ (HCT116) in L15 or McCoy's medium (cat. nos. 11415064 and 16600082; Gibco; Thermo Fisher Scientific, Inc.), respectively, with 10% fetal bovine serum (FBS; cat. no. S1860; Ultra-low Endotoxin; Biowest USA). Cells were split twice per week. Bovine-derived EVs were depleted from FBS by centrifugation at 100,000 × g for 4 h at 4°C (Sorvall^™ WX+ Ultracentrifuge with T-1270 rotor; Thermo Fisher Scientific, Inc.). PBS used in TEVs isolation was filtered (0.22 µm; Merck KGaA) and its purity was tested by nanoparticle tracking analysis (NTA; NanoSight LM10HS equipped with the LM14 488 nm laser module; Malvern Instruments, Ltd.). Cell lines were tested every month for Mycoplasma sp. contamination using a Mycoplasma PCR Detection kit according to manufacturer's instructions (cat. no. G238; Applied Biological Materials, Inc.). Supernatants from well-grown (confluency >90%) cell cultures were collected and spun at 500 × g for 5 min at room temperature (RT), and then at 3,200 × g for 12 min at 4°C to remove cell debris. The supernatants were again centrifuged at 100,000 × g for 2 h at 4°C. Pellets were washed in PBS to remove FBS and resuspended in filtered PBS. Quantification of TEVs was performed by NTA and protein measurement by the Bradford method (cat. no. 5000201; Bio-Rad Laboratories, Inc.). TEVs were tested for endotoxin contamination by Limulus test according to the manufacturer's instructions (cat. no. A39553; Pierce^™ Chromogenic Endotoxin Quant kit; Thermo Fisher Scientific, Inc.), and stored at −80°C.

Anticoagulated citrate dextrose A-treated blood from healthy donors was purchased from the Regional Center of Blood Donation and Blood Therapy (Krakow, Poland; agreement no. DZM/SAN/CM/U-678/2015; Bioethical Committee of the Jagiellonian University, Kraków, Poland; approval no. 1072.6120.1.2020). Human peripheral blood mononuclear cells were isolated from blood by standard Ficoll/Isopaque (cat. no. 17-1440-03; GE Healthcare) density gradient centrifugation (30 min, 800 × g, RT). Monocytes were separated from mononuclear cells by counterflow centrifugal elutriation with a JE-5.0 elutriation system equipped with a 5-ml Sanderson separation chamber (Beckman Coulter, Inc.), as previously described (21). Monocytes were suspended in RPMI-1640 culture medium supplemented with L-glutamine (cat. no. 11875093; Gibco; Thermo Fisher Scientific, Inc.) and gentamicin (cat. no. P06-13021; 50 µl/ml; PAN-Biotech GmbH). The purity of isolation was ≥95%, determined by staining with an anti-CD14 monoclonal antibody (cat. no. 555399; mAb; clone no. M5E2; BD Pharmingen; BD Biosciences). Monocytes were incubated with anti-CD14 for 30 min at 4°C in the dark (antibody concentration according to the manufacturer's protocol) and then washed with PBS, collected by flow cytometry (BD FACSCanto^™; BD Biosciences) and analyzed by FACSDiva Software (version 8.0.1; BD Biosciences).

To determine the expression of surface markers on monocytes by flow cytometry, the following mAbs were used: FITC anti-human CD44s (cat. no. 347943; clone no. G44-26), APC anti-human CD14 (cat. no. 555399; clone no. M5E2) and PE anti-human CD16 (cat. no. 555407; clone no. 3G8) (BD Pharmingen; BD Biosciences). Monocytes were incubated with anti-CD44, -CD14 and -CD16 for 30 min at 4°C in the dark and then washed with PBS as aforementioned. A total of 10,000 cells per run were analyzed on a BD FACSCanto^™ Flow Cytometer (BD Biosciences) using Diva Software (version 8.0.1; BD Biosciences). The monocyte subsets gating strategy is presented in Fig. S1. CD44 is used throughout the manuscript in the sense of CD44s, unless otherwise noted. CD44 expression was analyzed as mean fluorescence intensity (MFI) because the percentage of cells that exhibited fluorescence in every subpopulation was ~100%. CD44 expression on monocytes was evaluated after 2 and 18 h of culture (control), as well as after incubation with TEVs (TEV:monocyte, 5,000:1) after the same times, at 37°C.

The size and concentration of TEVs were defined by NTA. A suspension of 1,000 times diluted TEVs was loaded into the measuring chamber. The movement of particles was recorded in triplicates of 1-min videos (Fig. S2), after which the concentration, average size and mode values of TEVs were calculated. TEVs membrane structure was confirmed by MEMGlow staining, as previously described (22). MEMGlow solution (cat. no. MG01-02; 20 µM; Cytoskeleton, Inc.) was diluted in filtered PBS to 0.1 µM concentration, added to 40X diluted TEVs suspension and incubated for 30 min at RT in the dark. It was later analyzed by flow cytometry without washing.

EV markers were detected by western blotting (WB). Briefly, protein concentration in EV samples was determined by the Bradford method. Next, 20 µg/lane EV proteins extracted with Mammalian Protein Extraction Reagent (cat. no. 78501; Thermo Fisher Scientific, Inc.) were heated with loading buffer at 75°C for 10 min (cat. no. NP0007; 4X sample buffer; and cat. no. NP0004; 10X sample reducing agent; Invitrogen, Thermo Fisher Scientific, Inc.). Electrophoresis was performed at 180 V for 45 min on 14% polyacrylamide gel. Proteins were transferred onto a polyvinylidene fluoride membrane with semi-dry transfer at 25 V for 1 h, blocked at RT for 1 h with 1% bovine serum albumin in Tris-buffered saline with 0.1% Tween and incubated overnight at 4°C with rabbit anti-CD9 (cat. no. 13174S; clone no. D801A), anti-β-actin (cat. no. 8457S; clone no. D6A8) and mouse anti-ALIX mAb (cat. no. 2171S; clone no. 3A9) (Cell Signaling Technology, Inc.) diluted 1,000 times. Next, the membrane was incubated with secondary anti-rabbit (cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) or anti-mouse (cat. no. 31430; Invitrogen; Thermo Fisher Scientific Inc.) antibodies conjugated with horseradish peroxidase and diluted 2,000 times for 1 h at RT. The protein bands were visualized with SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (cat. no. 34578; Thermo Fisher Scientific, Inc.) by ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.).

The expression of CD44 and CD44v6 on TEVs was determined by flow cytometry with mAbs: FITC anti-human CD44 and PE anti-human CD44v6 (cat. no. 566803; clone no. 2F10). TEVs were incubated with CD44 and CD44v6 for 30 min at 4°C in the dark and analyzed on a BD FACSCanto^™ Flow Cytometer (BD Biosciences).

TEVs suspension was incubated with SYTO RNASelect (cat. no. S32703; Thermo Fisher Scientific, Inc.) at 37°C for 20 min in the dark. Excess dye was removed with Exosome Spin Columns (cat. no. 4484449; Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The effectiveness of labeling was determined by flow cytometry (FACS Canto II; BD Biosciences) in the FL1 channel (Fig. S3). SYTO RNASelect-labeled TEVs were termed SYTO RNA-labeled TEVs.

HA concentration in cell culture supernatants, TEVs and culture supernatants after TEVs isolation was measured by Quantikine ELISA Hyaluronan kit (cat. no. DHYAL0; R&D Systems, Inc.) according to the manufacturer's protocol. Briefly, samples were diluted 20–50-fold, depending on the HA content. The optical density was determined with ELX800NB, Universal Microplate Reader (450 nm; BioTek Instruments, Inc.). The concentration of HA was calculated by linear standard curve. The minimum detectable dose of HA was 0.068 ng/ml.

SYTO RNA-labeled TEVs were incubated with monocytes at a ratio of 1,000:1; 5,000:1 and 10,000:1 for 30 min, 2 h and overnight at 37°C. Following incubation, cells were washed and analyzed by flow cytometry. The binding of SYTO RNA-labeled TEVs to monocytes was determined by analysis of green fluorescence intensity and percentage of positive cells. Vital dye trypan blue was used for quenching extracellular fluorescence, as previously described (23). Briefly, 100 µl cell suspension was mixed with trypan blue solution (ratio, 1:1; final concentration 0.25 mg/ml), and reanalyzed by flow cytometry after 5 min. The fluorescence of monocytes incubated with SYTO RNA-labeled TEVs was compared with control monocytes that were not incubated with TEVs. The autofluorescence of TEVs was negligible.

Monocytes were stained with anti-CD14 and anti-CD16 antibodies, washed and incubated with SYTO RNA-labeled TEVs (TEV:monocyte, 5,000:1) for 15 min, 30 min or 2 h at 37°C. Excess TEVs were washed and cells were assessed by flow cytometry by analyzing the shift in green fluorescence intensity. Due to antibody labeling, the extracellular fluorescence quenching with trypan blue was not performed. MFI and the percentage of positive cells in the FL1 channel were determined for each gated subpopulation. To determine engulfment of TEVs, monocytes stained with mAbs were separated using FACS Aria II (BD Biosciences) into three populations: i) classical (CD14⁺⁺/CD16⁻); ii) non-classical (CD14⁺/CD16⁺⁺) and iii) intermediate (CD14⁺⁺/CD16⁺). Sorted cells were collected into polystyrene tubes and incubated with SYTO RNA-labeled TEVs (TEV:monocyte, 5,000:1). The experiments were performed only with HA-rich TEVs_HCT116 and limited to the shorter contact time of either 15 min or 1 h due to the small number of sorted non-classical and intermediate monocytes. The engulfment of TEVs was analyzed by quenching extracellular fluorescence with trypan blue; data are presented as a percentage of fluorescence-positive cells containing TEVs.

The role of CD44 in TEVs endocytosis was investigated by blocking this receptor on monocytes. A total of 2×10⁶/ml monocytes were incubated with anti-CD44 mAb (cat. no. BMS113; clone no. SFF-2; 10 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc) or appropriate IgG1 isotype control (cat. no. 14-4714-82; 10 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at 4°C. Monocytes were washed with RPMI, and incubated with SYTO RNA-labeled TEVs (TEV:monocyte, 5,000:1) for 30 min to 18 h at 37°C. The binding of SYTO RNA-labeled TEVs was determined by flow cytometry. To distinguish surface-bound and internalized TEVs, the quenching of extracellular fluorescence signals by trypan blue was used. MFI value of control monocytes (without TEVs) was subtracted from the MFI of monocytes incubated with SYTO RNA-labeled TEVs (elimination of autofluorescence effect), then the MFI of monocytes incubated with SYTO RNA-labeled TEVs without blocking CD44 was established as 100% and relative to this value, the percentage change of MFI (%MFI) for monocytes with blocked CD44 or with isotype control was calculated.

In the case of monocyte subsets, binding of TEVs to monocytes for 15 min or 1 h was determined by the percentage of fluorescence-positive cells (FL1 channel) for each gated subpopulation. The engulfment of TEVs was analyzed in sorted subsets after trypan blue treatment and presented as the percentage of fluorescence-positive cells.

Statistical analysis was performed by Statistica v. 13.3 (TIBCO Software Inc.). Mann-Whitney U, Wilcoxon signed rank, Student's t-test or Welch's test were used. For multiple group comparisons, one-way ANOVA followed by Tukey's post hoc test was used. Graphs were constructed in GraphPad Prism (version 8.0.1; Dotmatics). Detailed information about the statistical tests and the number of experiments is included in the respective figure legends. P<0.05 was considered to indicate a statistically significant difference.

TEVs_HCT116 and TEVs_SW1116 were isolated from cell culture supernatants by ultracentrifugation. The size distribution of TEVs was similar between TEVs_HCT116 and TEVs_SW1116 (Fig. 1A and B). Both tested TEVs expressed CD9 and β-actin as shown by WB. The expression of Alix was limited to TEVs_SW1116 (Fig. 1C). The MEMglow-positive fraction represented ~90% of both tested TEVs (Fig. 1D). The TEVs derived from HCT116 cells were significantly enriched in HA compared with those derived from SW1116 cells (200.43 vs. 0.26 ng/1×10¹¹ TEVs; n=13; Fig. 2A) which was associated with HA content in cell culture supernatants (Fig. 2B; n=4). The comparison of HA levels in cultured supernatants before and after TEVs isolation indicated that HA was primarily present in soluble form. HA detected in TEVs represented only 15 and 26% of the total HA in the culture supernatants of HCT116 and SW1116 cells, respectively (Fig. 2C; n=2). There were at least two forms of CD44 (CD44s and CD44v6) present on TEVs_HCT116 (~42 and ~58%, respectively) and TEVs_SW1116 (~32 and ~44%, respectively) (data not shown).

To investigate the role of CD44 in interaction of monocytes with TEVs, expression of CD44 on monocytes and their subsets was determined by flow cytometry (Fig. 3A). The three monocyte subsets, classical CD14⁺⁺/CD16⁻, intermediate CD14⁺⁺/CD16⁺ and non-classical CD14⁺/CD16⁺⁺, were determined based on expression of the CD14 and CD16 markers, as described by Ziegler-Heitbrock (16). Since all monocytes are CD44⁺, to assess differences in CD44 expression between subsets, MFI was evaluated in comparison to the appropriate isotype control (Fig. 3B). The highest MFI of CD44 was observed in the intermediate subpopulation and only the difference between intermediate and non-classical monocytes was statistically significant (n=23). The expression of CD44 on monocytes was not affected by incubation with TEVs (data not shown) for 2 and 18 h, however, an increase in CD44 MFI was observed after 2 and 18 h of culture (Fig. S4).

TEVs were labeled with green fluorescent SYTO RNA Select dye. The labeling did not affect the size distribution and concentration of TEVs (Fig. S2). The efficiency of TEVs labeling was high and controlled by flow cytometry (Fig. S2). TEVs were incubated with monocytes for 30 min and 2 and 18 h, and the attachment of TEVs to monocytes was analyzed by flow cytometry. More than 90% of monocytes was fluorescence-positive after 30 min incubation with TEVs from both cell lines, thus analysis of MFI shift was considered more informative than percentage of positive cells. The increase in monocyte MFI was associated with the number of TEVs/monocyte and exposition time (Fig. S5; n=3). A TEV:monocyte ratio of 5,000:1 was chosen for further experiments. The kinetics of TEVs attachment to monocytes are shown in Fig. 4. MFI of monocytes interacting with TEVs_HCT116 increased in a shorter time compared with TEVs_SW1116. This observation may indicate higher, statistically significant efficiency of attachment of TEVs derived from HCT116 cells compared with that of SW1116 cells (n=10; P<0.001). The percentage of monocytes showing fluorescence after 30 min incubation with TEVs_HCT116 was 5 times reduced after quenching with trypan blue (Fig. 5A). This suggested that approximately 20% of monocytes exhibited TEVs in the cytoplasmic area after that time. After 2 h incubation, ≥50% of monocytes engulfed TEVs. The internalization of TEVs_SW116 was not effective, as the uptake of TEVs after 30 min was ~3%. After 2 h, ~37% of monocytes contained TEVs_SW1116 (Fig. 5B). The internalization outcome/level of TEVs_HCT116 and TEVs_SW1116 after overnight incubation was comparable; in ~80% of monocytes, TEVs were located inside (Fig. 5; n=6).

TEVs carry HA and are effectively engulfed by monocytes, which is associated with high expression of major HA receptor CD44. To evaluate the role of CD44 in engulfment, control monocytes and monocytes with blocked CD44 molecules were incubated with SYTO RNA-labeled TEVs. MFI of green fluorescence channel was used for quantification of TEVs attachment and engulfment following trypan blue treatment. The attachment of TEVs decreased following CD44 blocking, however this was not significant (not shown). In turn, blocking of CD44 molecules on monocytes reduced TEVs_HCT116 engulfment by 50.75% compared with that of the isotype control (P=0.00739) after 30 min incubation with TEVs; after 2 and 18 h, the reduction was 1.8 and 22.75%, respectively, but this was not significant (Fig. 6A). For TEVs_SW1116, the CD44 blocking with the short time of incubation (30 min) was skipped due to the lack of engulfment; almost all fluorescence was derived from outside of monocytes (Fig. 5B). Blocking of CD44 molecules, followed by 2 or 18 h incubation with TEVs resulted in ~27 and ~6% reduction in the % MFI, respectively, compared with the isotype control. However, the differences were not statistically significant (Fig. 6B). Isotype control did not significantly diminish TEVs engulfment compared with control monocytes (n=4).

To determine whether differences in CD44 expression on monocyte subsets affect their interactions with TEVs, MFI value of subsets was analyzed after incubation with SYTO RNA-labeled TEVs. The highest increase in MFI was observed in the intermediate subset of monocytes, which may suggest that TEVs preferentially adhere to them (Fig. 7; n=5). This observation was similar for both types of TEVs; however, MFI after incubation with TEVs_SW1116 was lower (Fig. 7), which in turn, corroborates results obtained in previous experiments (Figs. 4 and S5). Next, the percentage of monocytes in subsets that showed green fluorescence after incubation with SYTO RNA-labeled TEVs was analyzed. Following 15 min incubation with TEVs_HCT116, ~46% of classical, ~29% of intermediate and ~20% of non-classical monocytes showed green fluorescence (Fig. 8A). In the case of TEVs_SW1116, the percentages of monocytes showing green fluorescence were 7.7, 12.5 and 7.5% for classical, intermediate and non-classical monocytes, respectively (Fig. 8B). The percentages of monocytes showing green fluorescence increased over time (n=3). Blocking of CD44 on monocytes resulted in a decrease in TEVs_HCT116, but not TEVs_SW1116 binding in all three subsets by 10.1, 9.94 and 8.63% for classical, intermediate and non-classical monocytes, respectively, however, the differences were not significant and observed only after 15 min of exposure to TEVs (Fig. 8). To exclude competition between subsets of monocytes in TEVs attachment/engulfment, experiments were repeated on sorted cells. TEVs were attached and internalized by all three subsets of monocytes. After 15 min contact, the differences in TEVs attachment were slight (Fig. 9A), with a non-significant predominance of intermediate monocytes. Over time, higher binding of TEVs was observed in classical monocytes compared with that in other subsets (91 vs. 60% for classical vs. intermediate monocytes and 91 vs. 54% for classical vs. non-classical monocytes (Fig. 9B). The highest engulfment of TEVs was observed in intermediate monocytes after 15 min of incubation (Fig. 9A); the percentage of positive intermediate cells was similar before and after trypan treatment which may suggest that TEVs were effectively internalized (47 vs. 43%, respectively). Blocking of CD44 resulted in a slight reduction in binding of TEVs to classical and intermediate monocytes and stronger to non-classical monocytes (decreased by 8, 13 and 27%, respectively). The internalization of TEVs was decreased following CD44 blocking; the effect was observed for all three subsets after 15 min and 1 h of contact, however this was not significant. The percentage of fluorescence-positive classical and intermediate monocytes with blocked CD44 receptors was reduced by ~42% after quenching of extracellular fluorescence. In non-classical monocytes, the decrease in the percentage of fluorescence-positive cells was ~13%.