General design of the study

This study aimed to investigate the potential of using WJ-Exo as carriers of the anticancer drug S3I-201 in treating TNBC. The research encompassed several key stages: characterization of the WJ-Exos through various analyses including TEM, SEM, DLS, flow cytometry, and Western blotting; loading of the exosomes with S3I-201 to create the nano-formulation WJ-Exo(S3I-201); assessment of the effects of WJ-Exo(S3I-201) on 4T1 cancer cells in vitro using a range of techniques such as MTT assay, flow cytometry, wound healing assay, Western blotting, and qPCR; and evaluation of the therapeutic efficacy of the nano-formulation in vivo using a tumor-bearing mouse model.

Reagents

RPMI-1640 medium (Gibco, NY, USA), Dulbecco’s Modified Eagle’s Medium F12 (DMEM F12) (Gibco, Grand Island, NY), fetal bovine serum (FBS) (Gibco, Grand Island, NY), anti-human antibodies labeled with fluorochromes against CD90, CD105, CD45, CD73, and CD14 (all from eBioscience), a Bicinchoninic Acid (BCA) kit (Thermo Fisher Scientific, USA), Exosome extraction kit (EXOCIB; Cibbiotech, Iran), Anti-CD63 (sc-5275, 1:200), anti-CD9 (sc-13118, 1:200), anti-CD81 (sc-166029, 1:100), and anti-β-actin (sc-47778, 1:300) (Santa Cruz Biotechnology, Dallas, Texas, USA). FITC-conjugated mouse anti-human CD63, APC-conjugated mouse anti-human CD9, and PE-conjugated mouse anti-human CD81 (BD, USA), S3I-201 (Sigma-Aldrich), MTT solution (5 mg/mL) (Sigma-Aldrich, USA), Annexin V‑PE/7‑aminoactinomycin D (7‑AAD) apoptosis detection kit (BD Biosciences, San Diego, CA, USA), specific antibodies (P-STAT3, STAT3, cleaved caspase-3, Bcl-2, and β-actin at 1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Following the incubation with an HRP-conjugated anti-rabbit secondary antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA), Trizol reagent (Roche Diagnostics GmbH, Mannheim, Germany), cDNA synthesis kit (Takara Biotechnology, Otsu, Shiga, Japan), SYBR-Green Master Mix (Ampliqon, Denmark).

Human WJ-MSC isolation

Anonymous umbilical cord units (15–20 cm) were obtained from postpartum women undergoing normal labor at Royan Institute (Tehran, Iran), focusing on individuals aged 20–40 years who delivered healthy infants (> 38 weeks). Informed consent was obtained from the tissue donors before using the samples. Under sterile conditions, UC units were freshly collected, cleaned with cold saline and antibiotics, and blood vessels were extracted to obtain Wharton’s Jelly [34] tissue. Subsequently, the WJ was mechanically cut into 1 cc pieces and evenly transferred into 75 cm² T-flasks (SPL Life Sciences, South Korea) filled with Dulbecco’s Modified Eagle’s Medium F12 (DMEM F12) (Gibco, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS) (Gibco, Grand Island, NY). Cultivation took place in a standard environment at 37 °C, 21% O₂ and 5% CO₂. After approximately one week, the culture medium was refreshed. After reaching decellularized WJ, the explants were removed and the cells were passaged at 80% confluence, showing fibroblast-like morphology. Routine observation, analysis and documentation of the MSCs at the morphological level were performed using an inverted light microscope (Nikon, Japan).

Characterization of human WJ-MSC

In accordance with the minimum criteria established by the International Society for Cell & Gene Therapy (ISCT) for the definition of human MSCs, the cells obtained were characterized. These criteria include plastic adherence, the expression of CD73, CD90 and CD105, the absence of hematopoietic and endothelial markers such as CD14, CD34 and CD45, and the ability to differentiate in vitro into adipocyte and chondrocyte lines [35].

Assessment of WJ-MSC adherence and surface antigen expression

The adherence of WJ-MSCs to standard culture plates and their morphological characteristics were examined using an inverted light microscope (Nikon, ECLIPSE TS100, Japan) with phase-contrast optics. The expression of specific surface antigens on WJ-MSCs was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were detached with trypsin-EDTA (0.05%) solution (Gibco, NY, USA) and WJ-MSCs were treated with anti-human antibodies labeled with fluorochromes targeting specific markers such as CD90, CD105, CD45, CD73, and CD14 (all from eBioscience) at 4 °C for 30 min in the absence of light. Isotype antibodies served as controls to exclude nonspecific binding, and FlowJo software (San Jose, CA) was used to analyze results.

Confirmation of multilineage potential of WJ-MSCs

The multilineage potential of the WJ-MSCs was confirmed by exposing them to adipogenic and chondrogenic induction media, staining with Oil Red-O and Alizarin Red-S (Sigma-Aldrich, Missouri, USA), respectively. 3 × 104 third passage MSCs were cultured in each well of 4-well plates under standard conditions (21% O2, 5% CO2, 37 °C, humidified incubator) for 20 days. Subsequently, the induction media were replaced by wells washed with phosphate buffered saline (PBS), fixed and stained. Cells that had undergone differentiation were examined using a reversed light microscope (Nikon, Japan) with phase contrast optics.

Preparation of WJ-Exo

WJ-MSCs-conditioning medium (WJ-MSCs-CM) was obtained from cells in passages 2 or 3. First, WJ-MSCs were cultured in 150 cm2 T-flasks (SPL Life Sciences, South Korea) under standard conditions (5% CO2, 37 °C). After reaching 70–75% confluence, the medium was gradually changed to a lower FBS content every 2 days and finally switched to FBS-free conditions. Cells were then cultured in serum-free medium for three days and the resulting CM was collected and filtered through a 0.22 μm membrane filter to remove debris.

Exosomes were isolated according to the manufacturer’s protocol (EXOCIB; Cibbiotech, Iran). In summary, the serum-free culture medium was centrifuged at 3000 rpm for 10 min at room temperature to remove debris. Reagent A was added to WJ-MSCs-CM at a ratio of 1:5, mixed by vortexing for 5 min and incubated overnight at 4 °C. After centrifugation at 3000 rpm for 40 min at 4 °C, the supernatant was discarded and the exosomes were resuspended in reagent B and then stored at -80 °C for further experiments.

Exosome protein quantification

A bicinchoninic acid (BCA) kit (DNAbiotech Co.) was used to quantify exosomal protein content. Bovine serum albumin (BSA) standards were serially diluted to prepare eight concentrations. Then, 25 µl of the BSA standards or samples were combined together with 75 µl of the BCA working reagent in a 96-well ELISA plate and mixed thoroughly. After incubating the plate at 60 °C for 60 min, the optical density (OD) was measured at 560 nm using an ELISA reader system (BioTek, USA).

WJ-Exosome characterization

WJ exosomes were characterized according to the MISEV2018 guidelines. This included morphological assessment (spheroid shape), sizing (30–150 nm) and expression of surface markers (CD9, CD63, CD81). Size and quality of the purified exosomes were assessed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). For TEM, exosomes were fixed in 1% glutaraldehyde (Sigma), placed on carbon-coated grids and examined with a LEO 906 transmission electron microscope (Carl Zeiss, Germany). The grids were prepared for TEM by washing with PBS, staining with 1% uranyl acetate and imaging with an Orius 200 camera with Digital Micrograph software (Gatan Inc, USA) at 80 kV.

Glutaraldehyde-fixed exosomes on glass slides were used for SEM analysis, which were observed with a TESCAN MIRA3 FEG-SEM after gold–palladium sputtering at 15 kV.

DLS was used to determine the size distribution of the purified exosomes after dilution in PBS (1:6 ratio) and then examined using the Zetasizer (Malvern, UK). The resulting data were analyzed with the Malvern software (Zetasizer Ver. 7.11).

The expression of surface markers (CD63, CD9 and CD81) was analyzed by Western blotting and flow cytometry using antibodies from Santa Cruz Biotechnology and FITC/APC/PE-conjugated antibodies from BD, respectively. For flow cytometry, markers were detected using the FACSCalibur flow cytometer and analyzed using FlowJo software (San Jose, CA).

For Western blotting, exosomes were heated to 95 °C for 5 min in a PMSF-containing lysis buffer [36] and resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). They were then blotted onto nitrocellulose membranes and blocked with non-fat milk powder in Tris-buffered saline with Tween 20 (TBST) for 1 h. The membranes were then probed overnight at 4 °C with primary antibodies. After washing with TBST, membranes were treated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit or anti-mouse antibodies for 1 h. Protein bands were detected using X-ray films and enhanced chemiluminescence (ECL) reagents.

Loading of S3I-201 into WJ-Exo

To load therapeutic cargo into WJ-Exo, the anticancer agent S3I-201 (Sigma-Aldrich) was incorporated into WJ-Exo. Purified exosome samples (100 µL) were suspended in 200 µL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride and 21% vol/vol OptiPrep). Different concentrations of S3I-201 (10, 100, 200, 300, 400, 500 µM) were combined with the exosome suspension at 4 °C and transferred to sterile electroporation cuvettes. Loading of the exosomes with S3I-201 was performed by applying a voltage of 600 V for 5 ms in 0.4 cm cuvettes using a multiporator (Eppendorf, USA). The mixture was then incubated at 37 °C for 30 min to facilitate recovery of the exosome membranes. The exosomes loaded with S3I-201 (WJ-Exo(S3I-201)) were isolated by centrifugation at 90,000 rpm for 1 h, and the loading efficiency of S3I-201 in exosomes was measured by HPLC-UV.

Determination of the S3I-201 load in exosomes

To determine S3I-201 loading in exosomes, drug loading in exosome studies was evaluated by HPLC. A standard curve for S3I-201 was first established using stock solutions of 10 mM S3I-201 in dimethyl sulfoxide (DMSO). S3I-201 standards from 1 to 500 µM (1, 10, 100, 200, 300, 400 and 500) were prepared with acetonitrile (MeCN). A C18 column (C18; 5 μm; 4.6 × 250 mm) (XBridge, Germany) with a mobile phase of H2O: acetonitrile (45:55, v/v) at 1 mL/min and 30 °C was used for the analysis and the absorbance was measured at 268 nm. The standard curve was constructed based on the area under the curve of the standards. For quantification of exosome active compounds, exosomes loaded with S3I-201 were combined with methanol (Merk, Germany), centrifuged at 13,000 rpm for 20 min at 4 °C, and the supernatant was evaporated with methanol. The solvent containing the exosomes loaded with S3I-201 was evaporated using a heating block (WiseTherm, Switzerland). The sample was then equilibrated with acetonitrile (Merk, Germany), sonicated for 25 min and injected (10 µL aliquots) into the HPLC system (Agilent 1200, USA).

Cell culture

Mouse breast cancer cells, 4T1 (NCBI code: C604, ATCC number: CRL-2539), were purchased from the Pasteur Institute in Tehran, Iran. These cells were cultured in RPMI-1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, NY, USA) and maintained at 37 °C in a 5% CO2 environment.

MTT assay

The cytotoxic activity of free S3I-201 and WJ-Exo(S3I-201) against 4T1 cells was determined using the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. First, 4T1 cells (1 × 104 cells/well) were cultured in a 96-well plate with complete media (RPMI-1640, 10% FBS and 1% antibiotic) for 24 h. Subsequently, 1 µg exosome with different concentrations of WJ-Exo (S3I-201) and free S3I-201 (10, 100, 200, 300, 400, 500 µM) were added to cell culture. After 48 h, MTT solution (5 mg/mL) (Sigma-Aldrich, USA) was added for 4 hours, followed by the addition of DMSO (Sigma-Aldrich, USA) to dissolve the formazan precipitate. Absorbance at 570 nm was measured using a spectrophotometer (Anthos, Austria) and cytotoxicity rates were determined in comparison to the negative control (DMSO only). All experiments were performed in triplicate and the IC50 values for loaded exosomes and free S3I-201 were calculated and compared using GraphPad Prism software.

Investigation of S3I-201cytotoxicity on PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from 7.5 ml of human whole blood by density centrifugation with a Ficoll gradient (Sigma-Aldrich, USA) at 850 g for 20 min at 20 °C, separating lymphocytes, monocytes and plasma. The layer containing the PBMCs was carefully removed from the original tube and transferred to a new 15 ml conical tube. The PBMCs were washed twice with 1× PBS containing 2% FBS, with each wash lasting 1 min. PBMCs were seeded at a density of 1 × 105 cells/well in 96-well plates with complete media (RPMI-1640, 10% FBS and 1% antibiotic) and treated with different concentrations of free S3I-201 (10, 100, 200, 300, 400, 500 µM). After a 48-hour incubation period, 20 µL of a 5 mg/mL MTT solution was added to each well, followed by 4-hour incubation at 37 °C. Optical density of samples evaluated by spectrophotometric analysis at 570 nm.

Apoptosis assay

Phosphatidylserine exposure on the plasma membranes of cells, indicative of apoptosis in 4T1 cells, was detected using the Annexin VPE/7aminoactinomycin D (7AAD) apoptosis detection kit (BD Biosciences, USA) according to the manufacturer’s instructions. 4T1 cells (5 × 105/well) were cultured in a 24-well plate with complete media for 24 h. Next, 5 µg WJ-Exo (S3I-201) loaded with 301.4 µM of S3I-201 (IC50 value) and 337.1 µM of free S3I-201 (IC50 value) were added to cell culture. This assay distinguishes between intact (Annexin V/7AAD), early apoptotic (Annexin V+/7AAD), late apoptotic (Annexin V+/7AAD+) and necrotic cells (Annexin V/7AAD+). In brief, cells were harvested after the treatment period with 0.25% trypsin, washed twice with cold PBS and resuspended in 100 µl 1X binding buffer. Addition of 5 µl Annexin VPE and 5 µl 7AAD followed by 15 min incubation at room temperature in the dark for 1 million cells. Subsequently, 400 µl of 1X binding buffer was added and flow cytometric analysis was performed using FlowJo software (Ashland, USA). Experiments were performed in triplicate.

Western blot analysis

4T1 cells (7 × 105 cells/well) were seeded in a six-well plate and allowed to grow to full confluence. Treatment medium consisting of free S3I-201 (337.1 µM), WJ-Exo(S3I-201) (5 µg exosome loaded with 301.4 µM of S3I-201), WJ-Exo or DMSO was then added to the cells. Protein extraction from 4T1 cells was performed using a cell lysis buffer (Cell Signaling Technology, USA). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated on 10% SDS–PAGE gels and transferred to PVDF membranes. The membranes were then blocked with 5% nonfat milk at room temperature and then probed with specific antibodies (P-STAT3, STAT3, cleaved caspase-3, Bcl-2 and β-actin at a dilution of 1:1000, Cell Signaling Technology, USA) overnight at 4 °C. After incubation with an HRP-conjugated secondary rabbit antibody (1:5000, Cell Signaling Technology, USA), the blots were visualized using Western ECL Blotting Substrates (Bio-Rad, USA).

Analysis of gene expression by real-time PCR

After 48-hour treatment of 4T1 cells (1 × 105 cells/96-well plate) with 1 µg of WJ-Exo(S3I-201, 301.4 µM), S3I-201 (337.1 µM), 1 µg of WJ-Exo and DMSO, total RNA, including miRNAs, was extracted with Trizol reagent (Gibco, Germany). RNA quantification was performed using NanoDrop (Thermo Fisher Scientific, USA) and 1 µg of total RNA was synthesized using a first-strand cDNA synthesis kit (Takara Biotechnology, Japan).

Quantitative RT-PCR was performed using the Step-One plus instrument (Applied Biosystems, USA) to determine the relative mRNA expression of Bax, Bcl-2, caspase-3 and β2-microglobulin (β2M). Target mRNA expression was evaluated in a 20 µL volume with Syber-green master mix (Ampliqon, Denmark), forward and reverse primers, and cDNA. The primers sequences were: forward primers (Bax: 5’-TGGCAGCTGACATGTTTTCTGAC-3’, Bcl-2: 5’-ATCGCCCTGTGGATGACTGAGT-3’, caspase-3: 5’-GGAAGCGAATCAATGGACTCTGG-3’ and β2M: 5’-ACTGAATTCACCCCCACTGA-3’), reverse primers (Bax: 5’-TCACCCAACCACCCTGGTCTT-3’, Bcl-2: 5’ -GCCAGGAGAAATCAAACAGAGGC-3’, caspase-3: 5’-GCATCGACATCTGTACCAGACC-3’ and β2M: 5’-AAGCAAGCAAGCAGAATTTGGA-3’).

Using β2M as the internal reference, relative gene expression in experimental and control groups was calculated employing the 2–ΔΔCT method, expressing results as fold regulation compared to the control. This analysis was conducted in three independent experiments, each performed in duplicate.

Examination of cell migration: scratch assay

4T1 cells (7 × 105 cells/well) were seeded in a six-well plate (SPL, South Korea) and allowed to grow to full confluence. Scratches were then made with a 1000-µl pipette tip and cells were washed with PBS, followed by PBS aspiration. The first images were taken at time 0 h. Treatment medium consisting of free S3I-201 (337.1 µM), WJ-Exo(S3I-201) (5 µg exosome loaded with 301.4 µM of S3I-201), WJ-Exo or complete DMEM control medium was then added to the cells. Microscopic images (Zeiss, 4 x magnifications) were taken after 24 h, 48 h and 72 h to ensure consistency of image position. Cell migration was measured using ImageJ software (National Institutes of Health, Bethesda, USA).

In vivo experiment

Female Balb/c mice aged 6–7 weeks were obtained from the Pasteur Institute in Tehran, Iran. Ethical approval for all animal experiments was obtained from the Institutional Animal Care and Use Committee of Semnan College of Medical Sciences, Iran (Ethic code: IR.SEMUMS.REC.1401.099). Mice were fed a standard laboratory diet and maintained under standardized 12/12-hour light/dark cycle conditions in an air-conditioned room with ambient temperature and humidity.

Establishment of xenograft tumors

4T1 cells were collected, suspended in PBS and injected into the flanks of female BALB/c mice weighing approximately 20 g at a concentration of 1 × 106 cells. To investigate the antitumor effect in vivo, the mice were randomly divided into four groups of 8 animals each when the tumor volume reached about 100 mm3 on day 10. The mice then received intraperitoneal injections of DMSO, S3I-201 (56 µg/dose), WJ-Exo (10 µg of exosome) and WJ-Exo (S3I-201) (10 µg of exosome loaded with 56 µg S3I-201/dose) on days 10, 12 and 14. The tumor volume was measured daily and the volume was calculated using the formula V = π × width × length × height/6. The general health, physical activity and body weight of the mice were monitored daily. The mice were monitored for signs of morbidity throughout the experiment. On day 31, 5 mice in each group were killed by cervical dislocation according to animal ethical guidelines, while 3 mice per group were retained for the assessment of survival rates. Tumor weights were determined and tumor lysates were prepared. The spleens were used after removal for splenocyte proliferation assays and determination of cytokine levels.

Preparation of tumor cell lysate

For the preparation of tumor cell lysate, the tumor tissue was divided into 1.5 ml microtubes and subjected to five freeze-thaw cycles alternating between liquid nitrogen and a 37 °C water bath. The extracted lysate was then stored at -80 °C for further use.

Isolation of mouse splenocytes

Mice were euthanized, and during dissection, fur and skin were disinfected with 70% alcohol. Careful manipulation was used to expose the spleen, which was then separated and placed in a 15-ml tube containing 2 ml of ice-cold PBS/FBS. The spleen and PBS/FBS mixture were transferred through a 70-µm cell strainer into a 50-ml tube. After trituration of the spleen without sectioning, 10 ml of PBS/FBS was added and the cell pellet was resuspended in 5 ml of ice-cold ammonium chloride hemolysis buffer. After incubation at 24 °C for 4 min, lysis was stopped with 10 ml ice-cold PBS/FBS and centrifugation at 400×g, 4 °C, for 10 min. The supernatant was discarded and the pellet was resuspended in 2 ml complete RPMI-1640 medium supplemented with 10% FBS and antibiotics. Splenocytes adjusted to a concentration of 1 × 106 cells/ml with viability above 90% (confirmed by trypan blue exclusion) were used for splenocyte proliferation assays and cytokine measurements in the cell culture supernatant.

Proliferation assay

Antigen-induced lymphocyte proliferation was measured by culturing splenocytes (4 × 105 cells/well) from mice in triplicate on 96-well plates for 48 h at 37 °C/5% CO2. Cultures were treated with tumor lysate (specific stimulator; 2.5 µg/ml) and PHA (non-specific stimulator; 10 µg/ml). A culture plate without stimulation served as a negative control. After further 12-hour incubation, the cultures were harvested and cell proliferation was measured using the MTT assay.

Measurement of cytokine production

After a period of 60 days following immunization, the animals were euthanized and the spleens were harvested to prepare a single cell suspension. These spleen cells were then cultured on 24-well plates at a density of one million cells/well. Cytokine levels were measured after 72 h in culture supernatants stimulated with PHA 5 µg/ml, tumor lysate at 100 µg/ml or in culture medium alone (negative control). Cytokine concentrations (IFN-γ, TNF-α, IL-4 and IL-1β) in culture supernatants were evaluated using the ELISA method, following the manufacturer’s instructors.

Statistical analysis

Data, presented as mean ± SD, underwent statistical analysis using One-Way and Two-Way ANOVA followed by Tukey post hoc test for multiple comparisons, along with Kaplan-Meier estimation, in GraphPad Prism 9.4 (GraphPad Software Inc, San Diego, CA, USA). Significance levels were denoted as *P < 0.05, **P < 0.001, ***P < 0.03, ****P < 0.0001 compared to the control group.