mDCs were obtained from DendroSURE™ Cell Culture Media (ExSURE, India), which induce the conversion of monocytes into mature dendritic cells within 48 h. THP1 cells were cultured in RPMI 1640 Medium (HiMedia) supplemented with 10% fetal bovine serum (FBS), 250 µg/mL amphotericin B, and 0.2% penicillin–streptomycin (Baxter et al., 2020). The cells were harvested by centrifugation at 800 rpm for 5 min, added to DendroSURE™ media, and cultured for 48 h to generate mDCs.

To package doxorubicin within the mDC-derived exosome, we cultured mDCs from DendroSURE™ media in the presence of doxorubicin (1.8 µg/mL) for 24 hrs. This led to the endogenous packaging of doxorubicin within the mDC-derived exosome. These doxorubicin-loaded mDC-derived exosomes are hereafter referred to as ExoDS.

Exosome isolation from mature dendritic cells (mDCexo) and doxorubicin-packed mDC-derived exosomes (ExoDS) was carried out using the exosome isolation reagent Exosure (ExSURE, India). The cell media were collected after 48 h of incubation of THP1 cells in DendroSURE™ media. After mature dendritic cell formation, mDCexo isolation was carried out. However, for ExoDS isolation, mDCs were supplemented with DendroSURE™ media and doxorubicin, and cell culture media were harvested after 24 h. The harvested media underwent centrifugation at 4,000 rpm for 30 min at 4°C, facilitating the removal of cellular debris. The resulting supernatant was carefully transferred to a fresh tube, and the appropriate volume of an exosome isolation reagent (Exosure) was added following the manufacturer’s instructions. This solution was meticulously mixed by vortexing and maintained at 4°C in an upright position for overnight incubation. The next day, the exosomes were precipitated by subjecting the mixture to centrifugation at 13,000 rpm for 1 h at 4°C. To ensure their long-term preservation, a suitable volume of 1× phosphate-buffered saline (PBS) was added to the pellet containing the exosomes, leading to their resuspension, after which they were stored at −20°C. The isolated mDCexo and ExoDS were characterized and confirmed by measuring their size, number, and expression of exosomal surface markers (Lai et al., 2022). Microscopic observation of the exosomes by transmission electron microscopy (TEM) was carried out, followed by dynamic light scattering (DLS) analysis (Malvern, United Kingdom) and nanoparticle tracking analysis (NTA) to determine the size and number of the exosomes in respective amounts of the solution. The molecular marker expression of exosomes CD9 and CD63 was checked by Western blot analysis (Huda et al., 2021).

For the breast tumor tissue and blood sample collection from the patient, we collaborated and obtained ethical committee approval from the Oncology Department at KIMS, Bhubaneswar (KIMS/SLRC/71/2022/01), and AIIMS Hospital (T/EMF/Surg.Onco/22/111). To isolate CTCs, a fresh 3-mL blood sample was obtained from breast cancer patients at KIMS and AIIMS hospitals in Bhubaneswar with applicable informed consent. PBMCs were derived from the blood sample by adding the Leucosure PMBC isolation reagent (ExSURE) or HiSep (HIMEDIA) in an equal volume, followed by density gradient centrifugation at 1,500 rpm for 45 min. The resultant buffy layer, rich in PBMCs, was carefully extracted and subjected to secondary centrifugation at 1,500 rpm for 10 min after adding an equal volume of 1× PBS. Subsequently, the isolated PBMCs were cultured in RPMI 1640 Medium supplemented with 15% FBS (Gibco), 0.2% penicillin–streptomycin, amphotericin B (250 μg/mL), insulin (9.5–11.5 mg/mL), epidermal growth factor (EGF), B27, and hydrocortisone (1,000 μg/mL) at 37°C with 5% CO₂. After an incubation period of 4 days, we observed the attachment of CTCs to the flask surface in 4 of the 13 samples collected from breast cancer patients (Koch et al., 2020).

Breast tumor tissue and the corresponding normal tissue specimens were procured from KIMS, Bhubaneswar (KIMS/SLRC/71/2022/01), and AIIMS Hospital (T/EMF/Surg.Onco/22/111) from a consenting patient. The tumor tissue was meticulously washed with 1× PBS and subsequently fragmented into minute segments using a sterile scalpel. To obtain single-cell suspension, the tumor tissue fragments were subjected to enzymatic dissociation with collagenase (1 mg/mL, prepared in serum-free RPMI 1640 Medium) overnight at 37°C in an orbital shaker at 80 rpm. After enzymatic digestion, the resultant tumor tissue suspension was passed through a nylon mesh. The single-cell suspension was centrifuged at 3,000 rpm for 5 min, and the cell pellet was thoroughly washed with 1× PBS to ensure the removal of unwanted debris and substances. The isolated cancer cells were then cultured in RPMI 1640 Medium supplemented with 15% FBS (Gibco), 0.2% penicillin–streptomycin, amphotericin B (250 µg/mL), insulin (9.5–11.5 mg/mL), EGF, B27 supplement, and hydrocortisone (1,000 µg/mL). The cells were cultured for 6–7 days with specific medium changes within a controlled environment, observed regularly for tumor cells, and utilized for further study (Chen et al., 2023).

To obtain CSCs from breast cancer cell lines, MDA-MB-231, MDA-MB-468, and MCF7 were grown as monolayers, trypsinized, and seeded into ultralow-attachment plates (Corning Inc., NY, United States) at a density of 2.5 × 10⁴ cells per well. We cultured these cells in ultralow attachment in serum-free RPMI 1640 Medium (HiMedia, India), supplemented with insulin (Sigma), EGF (BioLegend), and B27 (Sigma). The cells were cultured for 4–5 days for mammosphere formation (Kai et al., 2015). The primary spheres were subjected to secondary-sphere formation by dislodging the spheres using trypsin, followed by reseeding the single-cell suspension in mammosphere-specific media at 2.5 × 10⁴ cells per well into ultralow-attachment plates. To characterize the CSCs, we conducted CD44/CD24 analysis. Our analysis showed that >85% of the cells are CD44^+/CD24^–. We also conducted flow cytometric analysis of Nanog, Sox2, Oct4, ALDH1A1, ABCG2, and MRP1. The data are given in Supplementary Material.

To understand the effect of ExoDS in inducing apoptosis, we cultured breast cancer cells (MDA-MB-231, MDA-MB-468, and MCF7) and cancer stem cells derived from the mentioned cell lines and treated them with ExoDS and free Dox. The percentage of Annexin V-positive cells was determined by flow cytometry (Kabakov and Gabai, 2018).

After the complete treatment of THP1 cells with DendroSURE media, the cells were harvested, washed, and dissolved in 50 µl of 1× PBS. For the detection of differentiation and maturation in DCs, the cells were stained with fluorescent dye-conjugated mAb against selected markers (anti-CD14, anti-CD40, anti-CD83, and anti-CD86) (Beckman Coulter). The percentage of positive cells was evaluated using the CytoFLEX LX Flow Cytometer (Beckman Coulter, United States), and data were analyzed using CytExpert software (Lieschke et al., 2022).

For tagging ExoDS with CFSE dye, we labeled THP1 cells with the CFSE dye as per the instructions given (CellTrace™ CFSE Cell Proliferation Kit). Following the tagging of THP1 cells, the tagged cells were seeded in DendroSURE Cell Culture media for 48 h. Following incubation, the conditioned media were harvested, and the labeled exosomes were isolated using the Exosure exosome isolation reagent.

For tagging ExoDS with 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide (DiR) dye, the purified exosomes were labeled with the fluorescent dye, DiR, DilC18 (Invitrogen, United States and Canada, D-12731). The exosomes were incubated with DiR for 30 min at 37°C in the dark. After incubation, the exosome isolation reagent was added and incubated at 4°C overnight for labeled exosome isolation. After centrifugation at 13,000 x g for 1 h, the green pellets of the labeled exosomes were re-suspended in 1× PBS. The labeling of exosomes was tested at an excitation wavelength of 754 nm and an emission wavelength of 778 nm using the IVIS Spectrum Imaging System, and the exosomes were tracked after injection in mice at a specific interval of time (Santelices et al., 2022).

The cells were seeded on 12-mm-diameter coverslips. Following the differentiation of THP1 cells to mDCs after 48 h, the cells were fixed with 4% paraformaldehyde (PFA) (EMS) for 15 min at room temperature (RT). Primary and secondary antibodies were successively incubated for 1 h each at RT in PBS containing 0.2% BSA. The cells were permeabilized in 0.3% Triton X-100 + 5% BSA (in PBS) for 15 min at RT and blocked in 5% BSA (in PBS) for 30 min at RT; the primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at RT, both in blocking solution. Coverslips were then mounted on slides with DAPI (Invitrogen). Images were acquired on a Zeiss LSM 780 Confocal Microscope using a ×63 objective. At least 5 fields were captured to image a total of at least 10 cells per experiment (2 independent experiments). Image analysis was performed using ImageJ.

To study the toxicity of ExoDS on NOD/SCID mice (females), all the animals were purchased and housed in an animal house at the Institute of Life Sciences, Bhubaneshwar, India. The Institutional Animal Ethical Committee (Institute of Life Sciences, Bhubaneswar, India) approved the use of animals (Project no: ILS/IAEC-308-AH/NOV-22). All the methods associated with animal studies were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. MDA-MB-231 cells (1 × 10⁶) re-suspended in GFR Matrigel (Corning) were injected subcutaneously into the left mammary flanks of 6-week-old female NOD/SCID mice (n = 11). A total of eight (72.72%) of the injected mice developed subcutaneous tumors of palpable size by day 7 after tumor cell injection. For the toxicity study experiment, tumor-bearing mice were randomly assigned to four treatment groups (n = 2 in each group for a dose–response experiment): a) control exosome in PBS; b) ExoDS; c) free doxorubicin drug; and d) saline. The treatment was given every 48 h s for the next 12 days (6 dosages). The tumor volume and weight of the mice were calculated before every dose was administered. All the mice were sacrificed after 48 h of final treatment, and the subcutaneous tumors were excised along with the major vital organs (lung, kidney, heart, spleen, and liver) and normal tissue adjacent to the tumor site and stored in 10% formalin for further use (Warin et al., 2010).

The tumor volume of the mice was calculated using the following formula: Tumor Volume = Length of Tumor  x   Width   of   Tumor 2 / 2 .

The cell suspension of MDA-MB-231 cells and CSCs obtained from it and tumor tissues was suspended in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂ EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM Na₃VO₄) containing the protease inhibitor, followed by sonication (amplitude 35%, 5 s on and off for 10 min) and centrifugation (12,000 rpm, 15 min at 4°C). The protein was isolated and quantified using the Bradford assay. Equal amounts of protein were heated at 95°C for 10 min, resolved by 10% SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck). Then, the membranes were blocked with 5% BSA in 1× Tris-buffer saline with Tween 20 (TBST) and probed with specific primary antibodies overnight. The secondary HRP-conjugated goat anti-rabbit IgG antibody (ABclonal; 1:5,000) was used according to the manufacturer’s protocol. Antibody binding was detected using the chemiluminescence substrate and the ImageQuant LAS 500 System. The antibodies used included GAPDH (ABclonal; 1:2,000), beta-actin (ABclonal; 1:2,000), caspase-8 (ABclonal; 1: 1,000), CD63 (ABclonal; 1: 1,000), CD9 (ABclonal; 1:1,000), LC3A/LC3B (ABclonal; 1:1,000), and secondary antibody HRP-conjugated anti-rabbit IgG (ABclonal; 1:5,000) (Mishra et al., 2017).

The stemness marker gene (SOX2, OCT4, and NANOG) expression analysis was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was isolated from the MDA-MB-231 cells and tumor and normal tissue samples of mice using the TRIzol reagent (SRL, India) and quantified using a NanoDrop system. RNA was reverse-transcribed using the 5× PrimeScript RT Master Mix (Takara, Japan) in a total volume of 20 μL, and the resulting complementary DNA (cDNA) was used as a template for qRT-PCR (QuantStudio 5; Thermo Fisher). All reactions were carried out in triplicate using TB Green™ Premix Ex Taq™ II (Takara, Japan), and melt curve analysis was performed at the end of the PCR step (Iwahara et al., 2006). The Ct values of samples were normalized with the housekeeping gene GAPDH, and the fold change in transcript levels was estimated using the ΔΔCt method.

Primers used for the study are listed in the table.

The effect of ExoDS on the proliferation of MDA-MB-231 cells and MDA-MB-231 cell-derived CSCs was analyzed by flow cytometry. The cells were treated with four different sets: ExoDs, control exo, doxorubicin (5 ng/mL), and 1× PBS for 48 h. After incubation, the cells were harvested, washed, and dissolved in 50 µl of 1× PBS and stained with the Ki-67 proliferation marker antibody (BioLegend) for 30 min on ice (Yerushalmi et al., 2010). The percentage of proliferation was analyzed using the CytoFLEX LX System (Beckman Coulter, United States), and the proliferation index was calculated.

To carefully study the cytotoxicity of ExoDS and compare it with that of free doxorubicin and liposomal doxorubicin, we conducted an MTT assay on MDA-MB-231 cells. The viability of MDA-MB-231 cells treated with ExoDS, liposomal doxorubicin, and free doxorubicin was assessed after 48-h intervals using the MTT {3-(4,5-dimethylthiazol-2yl)−2,5-diphenyltetrazolium bromide} assay. MDA-MB-231 cells were initially seeded in triplicate into 96-well plates at 5 × 10³ cells/well in 200 µL RPMI 1640 Media and cultured to confluence over 1 day at 37°C and 5% CO₂. Upon reaching confluence, the cells were exposed to varying concentrations (0–500 ng/mL) of the aforementioned treatments. A 0.5 mg/mL MTT solution was prepared in 1× PBS, and 100 µL was added to each well after treatment, followed by 3-h incubation. After removing the supernatant, dimethyl sulfoxide (DMSO) was added to stop formazan formation. The 96-well plate was then shaken for 15 min in the dark at RT, and absorbance was measured at 570 nm (Maleki et al., 2020).

Values are shown as the standard error of the mean unless otherwise indicated. Data were analyzed, and the appropriate significance (p < 0.05) of the differences between mean values was determined using a two-tailed unpaired Student’s t-test in GraphPad Prism 7. Data are represented as the mean ± SEM.

To induce the maturation of THP1 cells into mDCs, we cultured THP1 cells with DendroSURE™ Cell Culture Media for 48 h. Our results showed that DendroSURE™ Cell Culture Media induced the differentiation of THP1 cells to mDCs within just 48 h (Figure 1A). We next analyzed the molecular signature of the induced mDCs. First, we observed a decrease in CD14 expression (a marker for monocytes) as monocytes differentiated to mDCs (Figure 1B). Next, flow cytometric analysis confirmed significantly enhanced expression of mDC markers like CD40, CD83, and CD86 (Figure 1C). To observe whether the mDCs generated are functional, we also carried out a FITC-dextran assay to observe the phagocytic capabilities of the generated mDCs (Figure 1D). Collectively, our results confirmed the generation of mature dendritic cells from monocytes within just 48 h of culture.

Next, we analyzed whether mDCs generated using DendroSURE™ Cell Culture Media secrete exosomes. To that end, we conducted an image analysis using CD63 and CD9 antibodies. Our results showed that mDCs produce exosomes after 48 h of culture with DendroSURE™ Cell Culture Media (Figure 2A). To load exosomes with doxorubicin, we cultured mDCs with DendroSURE™ and doxorubicin at a concentration of 1.8 μg/mL for 24 h. After 24 h, we harvested the condition media and isolated exosomes using the Exosure exosome isolation reagent. Following exosome isolation, we characterized the isolated exosomes. We first conducted DLS analysis (Figure 2B) and AFM analysis (Supplementary Figure S1) of the exosome to identify the size range of the isolated particles. To understand the shape of the isolated exosomes, we conducted a transmission electron microscopy (TEM) analysis (Figure 2C). We also conducted NTA to understand the number of particles isolated (Figure 2D). Lastly, to verify the molecular nature of the isolated exosomes, we analyzed key endosomal markers, including CD63 and CD9 (Figure 2E). To analyze the concentration of doxorubicin packaged with exosomes, we conducted fluorimetry (Figure 2F) and HPLC analysis (see Supplementary Material). Both methods confirmed that doxorubicin was packaged within mDC-derived exosomes, and the concentration was 0.2 ng/µL, showing ∼11% packaging efficiency. Since we used an endogenous drug-loading approach, where the mDCs were directly incubated with doxorubicin, we also analyzed whether mDCs incubated with doxorubicin showed any apoptosis following treatment with 1.8 µg/mL of doxorubicin for 24 h. To understand the same, we conducted an Annexin V assay and a Western blot analysis. Supplementary Figure S2 shows that mDCs incubated with doxorubicin for 24 h showed an ∼1.9-fold increase in Annexin V concentration (left panel); furthermore, the expression of caspase-8 and LC3 increased in mDCs after doxorubicin treatment (right panel).

To monitor the internalization potential of control exosomes derived from THP1 cells and ExoDS, we tagged THP1 cells and mature dendritic cells with the live cell-tagging CFSE dye. This led to the secretion of CFSE-tagged exosomes. ExoDS tagged with CFSE dye showed that it can be internalized significantly into cancer cells at 6 h (p < 0.0001) and cancer stem cells at 6 h (p < 0.0001) compared to control exosomes derived from the THP1 cell line, which enter at a much later time point of ∼12 h–24 h (Figure 3A). Next, to evaluate the cytotoxic effect of ExoDS on cancer cells, we conducted the MTT assay. We treated MDA-MB-231 cells with various concentrations of ExoDS, Lipo-Dox, and free doxorubicin for 48 h. Our results showed that ExoDS (IC₅₀ = 74.28 ng) is significantly more effective than liposomal doxorubicin (IC₅₀ = 164.11 ng) (p < 0.05) and free doxorubicin (IC₅₀ = 145.62 ng) (p < 0.05) in targeting cancer cells (Figure 3B). In order to understand the efficiency of ExoDS compared with the unconjugated form of doxorubicin in inducing apoptosis in cancer cells (Figure 3C) and cancer stem cells (Figure 3D) derived from MCF-7, MDA-MB-468, and MDA-MB-231 cells, we performed an apoptosis assay using Annexin V. Our analysis showed that ExoDS (5 ng/mL) induced almost comparable killing compared to the unconjugated form of doxorubicin (1.8 μg/mL) at an almost 400-fold lower concentration in MCF-7, MDA-MB-468, and MDA-MB-231-derived cancer cells and cancer stem cells. Furthermore, Ki-67 analysis also showed a significant (p < 0.05) decrease in the expression of Ki-67 following treatment with 5 ng/mL ExoDS compared to the same concentration of Lipo-Dox and free Dox (Supplementary Figure S3).

To check the efficiency of ExoDS in targeting cancer cells directly isolated from breast tumor tissue, we isolated tumor cells from breast cancer tissue and cultured them. Of the 13 tissues collected from KIMS Hospital, Bhubaneswar, we established sterile and stable primary cultures from 5 tumor tissues. The primary culture established from the five tissue samples was used next to test the efficacy of ExoDS. To that end, we treated the cells with 5 ng/mL ExoDS for 48 h, following which the cells were harvested and stained with the Annexin V antibody. Flow cytometric analysis confirmed that ExoDS caused significant apoptosis in tumor cells compared to the untreated control (Figure 3E). We also tested ExoDS on CTCs isolated from patient blood. CTCs were isolated and cultured as previously described. Of the blood samples collected from 13 tumor patients, CTC culture was established in 6 samples. The primary cultures of CTCs were treated with 5 ng/mL ExoDS for 48 h, after which the cells were harvested and stained with the Annexin V antibody. Similar to our previous observation, we found that ExoDS caused significant apoptosis in CTCs compared to the untreated control (Figure 3F).

To confirm whether ExoDS also induced apoptosis in healthy cells, we isolated and cultured PBMCs from the blood of healthy donors and breast cancer patients. Following the culture of PBMCs, we treated them with similar concentrations of ExoDS and free Dox. Our flow cytometric analysis of Annexin V-positive cells showed that free Dox induced significant apoptosis in PBMCs compared to the control, whereas ExoDS did not induce any significant apoptosis in PBMCs (Supplementary Figure S4; Figure 3G). Similar results were also observed in the immortalized HEK293 cells (Supplementary Figure S5). To further validate the effect of ExoDS on healthy cells, we isolated and cultured normal mammary cells from tumor-adjacent normal tissue. Of the six tumor-adjacent normal tissue samples that we received, a successful culture was established from three tissues. These cells were treated with 5 ng/mL ExoDS for 48 h, and the percentage of Annexin V-positive cells was observed by flow cytometry (Supplementary Figure S6). Data suggested that ExoDS failed to induce apoptosis in healthy cells. This proved that ExoDS is specific toward tumor cells and not healthy cells.

To validate the effect of ExoDS in vivo, we induced triple-negative breast tumors in NOD/SCID mice using MDA-MB-231 cells. Following tumor development, we randomized the mice into four groups, each having two mice: Dox, ExoDS, cont exo (THP1-derived exosomes), and saline. The Dox group was injected intravenously with 20 ng of Dox in 100 µL of 1× sterile-filtered PBS, and the ExoDS group was injected with the 20 ng equivalent of Dox in 100 µL of ExoDS solution. The cont exo group was injected with 100 µL of the cont exo solution. The treatment regimen continued for 4 weeks, with doses given at 48-h intervals.

We monitored the weight of each mouse and analyzed the tumor volume (in mm³) on days 1, 3, 6, 9, 15, and 20 (Figure 4A). Figure 4A shows a significant reduction in the increase in tumor volume following treatment with ExoDS compared to treatment with the unconjugated doxorubicin control. To confirm the distribution of ExoDS in vivo, whether it is targeted toward the tumor site or not, we intravenously injected DiR-tagged ExoDS to track their distribution across the entire body of the mouse. Figure 4B shows that our ExoDS is specifically targeted toward the tumor site at the right mammary fat pad, and it was localized into the tumor site within 12 h of injection. Furthermore, following the completion of the treatment regimen, we analyzed the DiR dye intensity in the tumor mass and the mammary fat pad tissue in the left mammary pad, where no tumor was induced. Figure 4C shows the localization of ExoDS specifically in the tumor site and not in the adjacent normal mammary fat pad tissue. We performed the Annexin V assay after extracting cells from the tumor mass (right mammary fat pad), healthy cells from the left mammary fat pad, and PBMCs isolated from mouse blood. Figure 4D shows that our ExoDS can induce significant killing in the tumor mass when compared to unconjugated doxorubicin at an equivalent concentration of 20 ng in 100 µL of 1× PBS. Furthermore, higher expression of the apoptotic marker caspase-8 and the autophagy marker LC3 was observed in ExoDS-treated tumor tissue by Western blot analysis (Supplementary Figure S7). Interestingly, qPCR analysis of stemness markers Nanog, Oct4, and Sox2 in ExoDS-treated tumor tissue revealed a significant reduction in the expression of stemness markers Nanog and OCT4 following treatment with ExoDS compared to free Dox (Supplementary Figure S7). Figure 4E shows that ExoDS has no toxic effect on the PBMCs isolated from the whole blood of mice in both groups when compared to unconjugated doxorubicin at an equivalent concentration of 20 ng in 100 µL of 1× PBS. Figure 4F shows that our ExoDS has no toxic effect on the cells isolated from the adjacent mammary fat pad without any tumor and induces no killing in the healthy mammary fat pad tissue when compared to unconjugated doxorubicin at an equivalent concentration of 20 ng in 100 µL of 1× PBS.