iRGD-modified 3D exosomes delivered miR-99b-5p induces ferroptosis to inhibit colorectal cancer progression by regulating FGFR3/PI3K/AKt pathway

Abstract

Background

Mesenchymal stem cells (MSCs)-derived exosomes present great potential as nanocarriers for targeted drug delivery. Moreover, the therapeutic efficacy of exosomes can be substantially enhanced through functional modifications and the incorporation of bioactive molecules.

Methods

In this study, the MSCs were cultured under two-dimensional (2D) and three-dimensional (3D) cell culture conditions. The culture supernatants were collected for isolating exosomes. The characteristics and yields of exosomes from 2D and 3D cultures were detected by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), western blot analysis, and bicinchoninic acid (BCA) assay. Subsequently, 3D exosomes were loaded with miR-99b-5p and modified with iRGD peptide were formed into a new engineered exosome, designated as iRGD-Exo-miR-99b-5p. The effects of these engineered exosomes on the progression of colorectal cancer (CRC) were assessed through a series of in vivo and in vitro experiments.

Results

The 3D-cultured MSCs exhibited a higher yield of exosomes and enhanced uptake by CRC cells. Further in vitro experiments demonstrated that 3D-exosomes loaded with miR-99b-5p effectively inhibit the proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of CRC cells. Results from a xenograft tumor model indicate that iRGD-modified exosomes were significantly enriched at tumor sites. Furthermore, exosomes modified with iRGD and loaded with miR-99b-5p were employed for CRC treatment, resulting in substantial tumor growth inhibition and enhanced the chemotherapy efficacy of 5-fluorouracil (5-FU) in vivo, without inducing notable toxicity or side effects. Mechanistically, exosome-mediated delivery of miR-99b-5p downregulated FGFR3 expression, thereby inhibiting the activation of the PI3K/AKt signaling pathway and promoting ferroptosis, ultimately attenuating CRC progression.

Conclusions

Collectively, iRGD-modified 3D exosomes loaded with miR-99b-5p were able to specifically target tumor sites, thereby significantly suppressing CRC growth through the induction of ferroptosis via regulating the FGFR3/PI3K/AKt signaling pathway. These findings suggest that functional engineering and bioactive loading of 3D-exosomes derived from MSCs represent a promising strategy for targeted cancer therapy.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-05129-8.

Introduction

CRC is one of the most common cancers worldwide, ranking third in terms of incidence and second in terms of mortality [1]. Despite progress in treatments like surgery and chemotherapy, advanced CRC prognosis remains poor due to challenges like systemic toxicity, drug resistance, and difficulty in penetrating the tumor microenvironment (TE) [2]. The TME, with its immunosuppressive cells and complex interactions, hampers treatment effectiveness, highlighting the need for innovative drug delivery systems that can bypass these barriers and reduce off-target effects.

In recent years, mesenchymal stem cell (MSC)-derived exosomes have gained recognition as promising nanocarriers for targeted drug delivery [3]. Compared to synthetic nanoparticles, MSC-derived exosomes possess numerous advantages: inherent biocompatibility and enhanced safety, minimal side effects, reduced immunogenicity, the ability to traverse biological barriers, high modification flexibility as well as enhancing targeting effects to selectively reach target cells or tissues [4, 5]. Notably, the advent of 3D culture systems has significantly improved exosome production by more accurately replicating the in vivo cellular microenvironment. In comparison to conventional 2D cultures, 3D cultures enhance the viability and yield of exosomes, alter cargo composition, including proteins, lipids, and nucleic acids, and improve therapeutic outcomes [6, 7]. These research advancements indicate that the exosomes derived from 3D-cultured MSCs can serve as an ideal carrier for delivering therapeutic agents to the tumor sites.

To enhance tumor-specific targeting, engineered exosomes functionalized with tumor-homing peptides, such as iRGD (a cyclic peptide binding to integrin αvβ3/5), have shown remarkable success. The iRGD peptide facilitates exosome penetration into deep tumor tissues by initially binding to integrin αvβ3/5 on endothelial cells, subsequently triggering neuropilin-1-mediated transcytosis [8]. Among the various cargo delivered by exosomes, microRNAs (miRNAs) have attracted considerable interest due to their regulatory roles in carcinogenesis and treatment resistance. Preclinical studies indicate that iRGD-modified exosomes loaded with therapeutic miRNAs exhibit increased tumor specificity and efficacy [9]. Our previous research confirmed that exosomal miR-99b-5p delivered by MSCs was able to limit the proliferation, invasion and migration of CRC cells in vitro, as well as suppressing tumor growth in tumor-bearing mice [10]. These findings highlight the potential of miRNA-loaded, iRGD-modified exosomes to overcome drug resistance and improve therapeutic precision. Consequently, this study aims to develop a novel therapeutic platform utilizing iRGD-modified exosomes derived from 3D-cultured MSCs for the targeted delivery of miR-99b-5p in CRC.

Materials and methods

Culture and identification of human bone MSCs

Human bone MSCs were purchased from Haixing Biosciences Co., Ltd in Suzhou, China. The cells were cultured in serum-free MSC medium (Dakewe Biotech Co, Shenzhen, China) containing EliteGro-Adv (Biomedical EliteCell Corp) and 1% penicillin-streptomycin. The cells were cultured in an incubator at 37 °C with 5% CO₂ and saturated humidity. For 2D culture, the cells were placed in cell culture dishes (Corning, NY, USA) and the medium was replaced every two days, and cell passage was conducted once the cells reached 80–90% confluence. The 3D suspension culture of MSCs was performed as previously reported [11]. Briefly, HBMSCs at passage 4 to 8 were seeded into ultra-low adhesion culture six-well plates (CLS3471, Corning, NY, USA) at a density of 4 × 10⁵ cells/well. The Live/Dead staining experiment for evaluating cell viability was detected using Calcein/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime, Shanghai, China) in accordance with the kit instructions. The cytoskeleton was stained with Actin-Tracker Green-488 (Beyotime, Shanghai, China). The stained cells were observed under an inverted microscope (Olympus, Japan). For the determination of the phenotypic markers of MSCs, the cells of 2D culture (passage 8) and the exfoliated cells from the 3D system were harvested, and surface antigen expression was detected by flow cytometry as previously reported [11]. In brief, the cells were incubated on ice in the dark with monoclonal antibodies containing CD14, CD45, CD73, CD90 and CD105 (Invitrogen, USA) for 20 min before flow cytometric analysis (Beckman Coulter, USA).

Isolation and purification of exosomes

Exosomes were isolated from conditioned media of MSCs cultured under 2D and 3D conditions. The two types of supernatants underwent a series of centrifugation steps at 4 °C: initially at 500 g for 10 min, followed by 2000 g for 20 min, then 12,000 g for 30 min, and subsequently filtered through a 0.22 μm membrane. This was followed by ultracentrifugation at 100,000 g for 70 min. The resulting pellets were washed with phosphate-buffered saline (PBS) and re-centrifuged at 100,000 g for an additional 70 min. Exosomes derived from both 2D and 3D cultures were resuspended in PBS for subsequent use. The presence of exosomal specific markers was examined via western blotting. The ZetaView particle tracker (Particle Metrix GmbH, Meerbusch, Germany) was used to assess the concentration and size distribution of the exosomes. For morphological analysis, the purified exosomes were adsorbed onto carbon grids and negatively stained with 2% uranium acetate for three minutes, followed by examination with TEM. Exosomal protein quantification was conducted by measuring absorbance at 562 nm using the BCA Protein Assay Kit (No. WB6501; NCM, Suzhou, China), in accordance with the manufacturer’s instructions.

CRC cell culture and cellular uptake assay

CRC cell lines (SW480 and LoVo) were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in 5% CO₂. To investigate the mechanisms involved in ferroptosis, cells were treated with the ferroptosis inducer Erastin (Cat. No. HY-15763, MCE), the ferroptosis inhibitor ferrostatin-1 (Cat. No. HY-100579, MCE, New Jersey, USA), the pyroptosis inhibitor Z-VAD-FMK (Cat. No. A834991, AmBeed, Shanghai, China), the RIP1 inhibitor Necrostatin-1 (Cat. No. A181851, AmBeed), the PI3K signaling pathway inhibitor LY294002 (Cat. No. HY-10108, MCE), and the PI3K signaling pathway activator 740Y-P (Cat. No. HY-P0175, MCE). For cellular uptake assays, the membrane fluorescent dye DiR (Aladdin Scientific Corp, Shanghai, China) was added into the exosome solution at a concentration of 20 µM. Following vortex mixing for one minute, the solution was incubated at 37 °C for 30 min. Subsequently, the samples were subjected to two washes with PBS and centrifuged at 100,000 g for 70 min. The DiR-labeled exosomes were then co-cultured with CRC cells at a cell-to-exosome ratio of 1:10000 for 24 h. The F-actin of the cells was stained in accordance with the instructions of the Actin-Tracker Green-488 kit (Beyotime), and the nuclei were counterstained with DAPI for visualization. The uptake of DiR-labeled exosomes by the CRC cells was then observed by confocal laser scanning microscopy (Zeiss, Germany).

Cell transfection

CRC cells were inoculated at a density of 2 × 10⁵ cells/well into a 6-well plate one day before transfection. Transfection was performed when the cells were 70–90% confluent. CRC cells were transfected with miR-99b-5p/NC mimic (RiboBio, Guangzhou, China), Si-FGFR3 and pcDNA3.1-FGFR3/control (Hanbio, Shanghai, China) plasmids, respectively. Lipofectamine 8000 Transfection Reagent (Beyotime, Shanghai, China) was used for all transfections by following the manufacturer’s instructions. The sequences are listed in Supplementary Table S1.

RNA isolation and quantification

Total RNA, including miRNA, was extracted from CRC cells or exosomes utilizing the Trizol reagent (No. 15596026CN; Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was conducted according to the PrimeScript RT Reagent Kit (No. RR036A; Takara, Beijing, China). Quantitative real-time PCR was performed using SYBR Green Master Mix (No. Q711-02; Vazyme, Nanjing, China) on an Mx3000P Real-time QPCR System (Analytik Jena, Carlsbad, Germany). U6 and GAPDH were set as internal standard. Primers for miRNA were procured from Gensysbio (Nanning, China). The primers for mRNAs were designed and synthesized by Gensysbio (Nanning, China), and the primer sequences are listed in Supplementary Table S2. The relative expression levels of target genes were calculated using the 2^−ΔΔCT method as previously described.

Western blot analysis

Cells or exosomes were lysed in radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors. Proteins were separated by SDS-PAGE gel electrophoresis and subsequently transferred onto PVDF membranes. Following a blocking step with 5% skimmed milk powder for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were listed in Supplementary Table S3. Secondary antibody detection was performed using HRP-conjugated Goat Anti-Rabbit IgG(H + L) (SA00001-2, 1:10,000; Proteintech). Detection of protein bands was achieved using an ECL reagent (P10060; NCM, Suzhou, China) and visualization was conducted with a C-DiGit Blot Scanner (UVP ChemStudio815, Beijing, China). Quantification of band intensities was performed using Image J software.

CCK8 assay

Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8; No. C6005; NCM), in accordance with the manufacturer’s protocol. Once the cells achieved 70–80% confluence, disperse the cells and inoculate them into 96-well plates at a density of 3 × 10³ cells per well. Incubation periods were set at 12, 24, 36, and 48 h, 10 µL of CCK8 solution was added to each well. After an additional 1.5 h incubation at 37 °C, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher, USA). Experiments were performed in triplicate wells and repeated at least three times independently.

Wound healing assay

For the wound healing assay, CRC cells (2 × 10⁵ cells/well) were seeded into six-well plates and cultured until a confluent monolayer was formed. A scratch was introduced using a 100µL pipette tip to simulate a wound, after which the cells were incubated in serum-free DMEM. Images of the wound area were captured at 12 and 24 h using an inverted optical microscope (Axio Vert.A1, Carl Zeiss AG, Oberkochen, Baden-Württemberg, Germany). Wound closure was quantified with ImageJ software.

Transwell assay

Cell invasive capacity of the cells was assessed utilizing Transwell chambers (Jet Bio-Filtration Co, Guangzhou, China) with 8 μm pore polycarbonate (PC) membranes. Matrigel (Corning, USA) was thawed at 4 °C overnight and subsequently diluted with a serum-free medium on ice. The upper chamber of the Transwell was pre-coated with 100 µL of diluent containing Matrigel and incubated at 37 °C for 2 h to solidify. Subsequently, cells were resuspended in serum-free medium at a density of 2.5 × 10⁵ cells/mL and introduced into the upper chamber. Concurrently, 600 µL of medium supplemented with 20% FBS was added to the basolateral chamber as a chemoattractant, and the assembly was incubated for 48 h at 37 °C with 5% CO₂. Non-invaded cells on the upper surface of the PC membranes were removed with a cotton swab. The invasive cells (on the lower surface of the PC membrane) were fixed with 4% paraformaldehyde solution for 20 min and subsequently stained with 0.1% crystal violet solution (Solarbio, Beijing, China) for 10 min at room temperature. The stained cells were visualized under an inverted microscope (Olympus, Japan). Invasive cells were quantified by counting stained cells in at least five random fields per PC membrane. Following this, the cells were incubated in a Transwell chamber not coated with Matrigel to assess cell migration.

Intracellular iron assay

To quantify total iron and Fe²⁺ levels in CRC cells and mouse tissues, the Iron Assay Kit (Cat. No. TC1015; Leagene, Beijing, China) was employed. According to the manufacturer’s instructions, ferrozine was added to the samples to release bound Fe³⁺. Subsequently, a reducing agent was introduced to convert Fe³⁺ to Fe²⁺, which then reacted with ferrozine to form a purple-colored complex. The absorbance of the resulting solution was measured at 562 nm using a microplate reader to determine total iron level.

Glutathione assay

According to the manufacturer’s instructions, the levels of GSH in CRC cells or mouse tumor tissues were measured using a GSH assay kit (Cat. No. T01036; Leagene, Beijing, China).

Lipid reactive oxygen species detection

The levels of lipid Reactive Oxygen Species(ROS)were determined using the ROS Assay Kit (Cat. Nos. CA1420 and CA1410; Solarbio, Beijing, China). For cell-based measurements, after the designated treatments, 5 µM of the red fluorescent probe was added to the culture medium, and cells were incubated at 37 °C for 30 min. The fluorescence signal was subsequently quantified using ImageJ software. For tissue-derived samples, freshly excised tumor tissues were first rinsed three times with 1× PBS, minced into small fragments, and washed again to remove residual blood and debris. Tissue digestion was carried out by adding an appropriate concentration of collagenase and incubating at 37 ℃ for 30 min, with gentle agitation every 5 min. The reaction was stopped by the addition of FBS-containing medium. Following centrifugation at 500 g for 5 min, the supernatant was discarded, and the cell pellet was washed twice with 1× PBS. The resulting suspension was incubated with diluted DHE probe (1:500) at 37 °C for 30 min. Fluorescence was measured on a microplate reader (excitation: 500 nm; emission: 610 nm). Protein concentrations were determined by diluting 50 µL of the tissue homogenate tenfold with PBS, and 100 µL of the diluted sample was used for quantification. Lipid ROS levels in tissue were expressed as relative fluorescence units (RFU) per milligram of protein.

Immunofluorescence (IF) assays

Cells were collected and seeded into small culture dishes, followed by overnight incubation. After three washes with PBS, the cells were fixed in 4% paraformaldehyde for 15 min and subsequently permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Non-specific binding was blocked by treating the samples with 5% bovine serum albumin (BSA) for 30 min. Primary antibodies against E-cadherin (Cat. No. CY1155, 1:200, Abways) and Vimentin (Cat. No. YM8324, 1:500, Immunoway) diluted in a universal antibody diluent were applied, and the cells were incubated at 4 °C overnight. On the following day, samples were washed and then incubated for 1 h at 20–37 °C with the appropriate fluorescent secondary antibodies (Cat. No. SA00013-2, 1:500, Proteintech). Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) for 3 min in the dark. Images were acquired using a Zeiss LSM 980 confocal laser scanning microscope equipped with Airyscan 2. Immunofluorescence quantification was performed on at least three independent experiments. For each sample, a minimum of 3 randomly selected microscopic fields were imaged using a fluorescence microscope at ×200 or ×400 magnification. Regarding the fluorescence quantification, the analysis was performed using ImageJ. The integrated density was measured for each region of interest (ROI). To normalize, we subtracted the background fluorescence and then divided the values by the nuclear area to account for differences in cell density. Data were averaged per field and then per sample to ensure statistical independence.

miR-99b-5p loading into 3D-exosomes

To enhance miR-99b-5p expression in MSCs derived 3D-exosomes, miR-99b-5p mimic was loaded into the exosomes with exosomal RNA loading kit (Echo Biotech, Beijing, China) following the instructions. Briefly, add an appropriate amount of ETP Dilution Buffer to the dry powder of the exosome-Transit Peptide to ensure complete dissolution. Sequentially, the miR-99b-5p mimic, 3D-exosomes, the ETP solution, and the reaction buffer with one-tenth the volume of the exosomes were added in sequence. The reaction mixture was then incubated at 37 °C in the dark for 2 h, with continuous shaking at 150 rpm. Next, free oligonucleic acids were removed by centrifugation at 4000 g for 20 min using a 100 kD ultrafiltration tube (UFC8100, Millipore). After washing with wash buffer for 2 times, resulting in the obtainment of miR-99b-5p loaded 3D-exosomes from MSCs (3D-Exo-miR-99b-5p).

Preparation of iRGD-modified exosomes

The 3D-exosomes derived from MSCs were modified with iRGD utilizing the ExoBrooch-iRGD exosomal modification lipid-anchor kit (Echo Biotech, Beijing, China) according to the manufacturer’s protocol. Initially, the dilution buffer was added to the iRGD-lipid-anchor to ensure complete dissolution. Subsequently, the iRGD-lipid-anchor solution, 3D-exosomes, and a reaction buffer amounting to one-tenth of the exosomes volume were sequentially combined. The reaction mixture was incubated at 25 °C for 3 h with continuous shaking at 250 rpm, followed by static incubation at 4 °C for 24 h. Unreacted substances and byproducts were then removed using a 100 kD ultrafiltration tube (UFC8100, Millipore) through centrifugation at 4000 g for 20 min. Ultimately, the iRGD-modified exosomes were obtained. These exosomes were labeled with FAM fluorescence, allowing for detection with excitation at 488 nm and emission between 515 nm and 535 nm. The labeled exosomes can be detected. The loading quantity was determined using the standard curve of the lipid-anchor targeting peptide.

Long-term safety assessment in vivo

Six BALB/c nude mice (5 weeks, weighing 17–21 g) were randomly divided into two groups (n = 3 per group) to evaluate the long-term systemic safety of iRGD-Exo-miR-99b-5p. The experimental group received two repeated doses of 5 × 10¹⁰ iRGD-Exo-miR-99b-5p via tail vein injection. The control group received an equal volume of PBS. The experimental procedures involving mice were carried out in a randomized order. All the mice were free to drink and eat and lived in a standard environment with suitable temperature and humidity control. Throughout the study, the mice were monitored for visible behavioral abnormalities or signs of toxicity, weighed every seven days, and euthanized one week following the final injection. The blood samples of mice were collected for hematological analysis. Automatic Biochemistry Analyzer (Raito, Shenzhen, China) was used to measure the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine (CREA) and blood urea nitrogen (BUN). Major organ tissues, including the heart, brain, liver, lung, spleen and kidney, were harvested and subjected to hematoxylin and eosin (H&E) staining, following established protocols. The work has been reported in line with the ARRIVE guidelines 2.0.

In vivo biodistribution and tumor-targeting of iRGD-modified exosomes

Four-week-old BALB/c nude mice (weighing 16–21 g) were obtained from the Animal Center of Guangxi Medical University and maintained under standard laboratory conditions. A LoVo cell suspension (210⁶ cells/mice) was subcutaneously injected into the right posterior flanks of each mouse. A total of six mice were randomly divided into two groups, each group containing three mice. When the tumor volume reached approximately 60 mm³, the mice were injected with DiR-labeled iRGD-Exo-miR-99b-5p or 3D-exosomes via tail vein. Tumor volume (mm³) was calculated with the formula: (lengthwidth²)/2. For in vivo imaging system (IVIS), mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (PS1714, SAITONG, Beijing, China) at a low dose (50 mg/kg, a dose that provides surgical anesthesia without lethality). The biodistribution of DiR-labeled exosomes in mice was recorded by an IVIS imaging system at 0, 12, 24, 36, and 48 h after injection. Beyond 48 h post-injection, the majority of exosomes are cleared and become undetectable. At the end of the study, the nude mice were euthanized through an intraperitoneal injection of pentobarbital sodium (200 mg/kg) followed by cervical dislocation. Subsequently, the tumors and the major organs were dissected and imaged for further analysis. The fluorescence intensity was then analyzed using an IVIS (PerkinElmer, USA).

H&E and immunohistochemistry (IHC) staining

Mouse tissues were dehydrated and fixed using 4% paraformaldehyde followed by an ethanol gradient, embedded in paraffin, and sectioned continuously at a thickness of 5 μm. For H&E staining, deparaffinized sections were stained with hematoxylin and eosin solutions, followed by air drying and mounting with neutral resin. For IHC staining, endogenous peroxidase activity was blocked, and antigen retrieval was performed. Sections were then incubated with goat serum for blocking, followed by sequential application of the primary antibody against Ki-67 (Cat. No. GB111499, 1:500; Servicebio, Wuhan, China) and an HRP-conjugated goat anti-rabbit secondary antibody (Cat. No. GB23303, 1:200; Servicebio, Wuhan, China). Staining was developed using diaminobenzidine (DAB), and sections were counterstained with hematoxylin. Quantitative analysis of staining signals was performed on three randomly selected fields per sample under a dual-observer light microscope (BX43, OLYMPUS, Tokyo, Japan).

Anti-tumor efficacy in vivo

The animal models were established in four-week-old nude mice as described above. When tumor volumes achieved about 100 mm³, the 20 tumor-bearing mice were randomly divided into four groups (n = 5 per group), as follows: Group 1: PBS; Group 2: 5-FU; Group 3: iRGD-Exo-miR-99b-5p; Group 4: iRGD-Exo-miR-99b-5p + 5-FU. Group sizes were determined based on data derived from our prior studies using similar tumor-bearing mice model, ensuring the minimum number of animals necessary to achieve statistically meaningful results [10]. 5-FU was administered intraperitoneally into the mice at a dose of 10 mg per kilogram of body weight. The exosomes of different treatments were tail-intravenously injected into mice at a dose of 510⁵ particles/mouse each time, once every five days, for a total of three times. Subsequently, the nude mice were euthanized and the tumor tissues were isolated, weighed, and photographed. Tumors were collected for H&E staining, IHC, western blot analysis, and detection of ferroptosis indices. Humane endpoints for the experiment include severe body weight loss, excessively large tumor burden, or ulceration and infection at the tumor site. No animals exhibited signs requiring early euthanasia during the study. All in vivo experiments were conducted in accordance with the ARRIVE guidelines 2.0.

Statistical analysis

All in vitro experiments were conducted in biological triplicate. Statistical analysis was performed with GraphPad Prism version 8.0 (La Jolla, CA, USA) and presented as mean ± standard deviation (SD). Two-tailed student’s t-test was used to examine significant differences between two groups, while one-way ANOVA was performed for comparisons involving more than two groups. P < 0.05 was considered statistically significant. Significant differences are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.

Results

Establishment of 3D culture systems and the characterization of 3D-MSCs

Schematic diagrams of 2D and 3D culture systems for MSCs are illustrated in Fig. 1A. In the 2D culture system, MSCs exhibit an adherent growth pattern and display a fibroblast-like morphology (Fig. 1B). Conversely, in the 3D culture system, spontaneous cell aggregation is observed, leading to the formation of near-spherical structures within 12 h of culture, which become increasingly dense after 24 h. During the initial 4–5 days of suspension culture, the diameter of these spheres progressively increases with time, and then the growth rate subsequently diminishes, coinciding with the emergence of new 3D spheres (Fig. 1C). The Live/Dead experiment was employed to assess the biological activity of MSCs in both 2D and 3D environments. The results showed that the green fluorescence of living cells was uniformly distributed in 3D cell spheres, and the dead cells that showed red fluorescence were barely detected under confocal microscopy (Fig. 1D). Fluorescence imaging of MSCs with stained cytoskeleton revealed that in 2D culture MSCs form dispersed, spindle-shaped fibroblast-like cell clusters, whereas in 3D culture, MSCs are closely connected, forming compact spheres (Fig. 1E). Flow cytometry analysis demonstrated that both groups of MSCs exhibited a high positivity rate (> 98.5%) for MSCs surface markers including CD105, CD73 and CD90, while showing a low expression (< 2%) for hematopoietic stem cell surface markers (CD45 and CD14) (Fig. 1F). Furthermore, expressions of MSCs surface markers remained consistent between cells cultured in 2D and 3D environments. This consistency suggests that 3D culture conditions preserve the fundamental characteristics of stem cells, with no alterations observed in their stem cell phenotype.

Fig. 1.

The establishment of 2D and 3D culture system of MSCs. A Schematic diagrams of 2D and 3D culture of MSCs. B, C Representative images of 2D culture of MSCs and 3D MSCs aggregates. D Representative live/dead images of 2D and 3D MSCs. E F-actin Immunofluorescence staining in 2D and 3D MSCs. F Surface markers of MSCs under 2D and 3D conditions were analyzed by flow cytometry

Isolation and characterization of MSCs-derived exosomes in 2D and 3D culture

Exosomes were isolated from the supernatants of MSCs cultured in 2D and 3D systems via ultracentrifugation. TEM assay revealed that exosomes derived from both 2D and 3D culture systems displayed a typical saucer-like double-layer structure with an approximately diameter of 100 nm (Fig. 2A). NTA indicated that the median particle sizes of exosomes from 2D and 3D cultures were 177.07 ± 9.73 nm and 168.03 ± 9.65 nm respectively, with no statistically significant difference observed (p > 0.05; Fig. 2B). Western blot assay confirmed the presence of the positive markers (TSG101, Hsp70 and CD81) and the absence of negative marker (Calnexin), thereby affirming the purity of the isolated exosomes (Fig. 2C). Quantification of the total protein yield using the BCA assay demonstrated that MSCs cultured in the 3D system produced a significantly higher protein yield compared to those cultured in the 2D system (Fig. 2D). Furthermore, fluorescent labeling of exosomes with DiR, followed by confocal laser scanning microscopy, revealed that CRC cells (LoVo and SW480) internalized a significantly greater quantity of exosomes derived from 3D-cultured MSCs than from those cultured in 2D (Fig. 2E–H). This finding suggests that 3D exosomes are more readily taken up by CRC cells. Therefore, 3D exosomes derived from MSCs were selected for further experiments.

Fig. 2.

Characterization of exosomes produced by MSCs in 2D and 3D spheroid culture. A The morphology of exosomes was observed by TEM. B The concentration and particle size distribution of MSCs-exosomes were determined by NTA. C Exosomal marker proteins (CD81, Hsp70, TSG101, and Calnexin) were detected by western blot assay. D BCA assay to determine total protein yield (n = 3). E–F Cellular uptake of DiR-labeled exosomes in CRC cells images by confocal microscopy. G–H The relative mean fluorescence intensity (MFI) of DiR-labeled exosomes in CRC cells (n = 3). Student’s t-test was used to compare two groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

The 3D-Exo-miR-99b-5p derived from MSCs suppressed the growth of CRC cells in vitro

The miR-99b-5p was encapsulated into exosomes derived from 3D-culture MSCs to create engineered exosomes, designated as 3D-Exo-miR-99b-5p. TEM assay demonstrated that the 3D-Exo-miR-99b-5p exhibited a typical cup-shaped morphology (Fig. 3A). As determined by NTA, the average diameter of 3D-Exo-miR-99b-5p was 175 ± 8.43 nm, with no significant differences from the particle size of unmodified 3D-exosomes (p > 0.05; Fig. 3B). Subsequent co-incubation of 3D-Exo-miR-99b-5p with CRC cells (LoVo and SW480), which were labeled with DiR, demonstrated that the exosomes could be internalized by CRC cells, leading to a significant increase in intracellular miR-99b-5p levels, as confirmed by real-time PCR (Fig. 3C–E). To further confirm that the detected miR-99b-5p originated from intact exosomal cargo, we pre-treated Exo-miR-99b-5p vesicles with RNase prior to co-culture. At the detection time points of 3 h, 6 h, and 18 h, RNase treatment did not significantly reduce miR-99b-5p levels in recipient cells (Figure S1A), indicating that this miRNA was protected within the exosomal lumen and delivered as functional cargo, rather than originating from surface-bound miRNAs or endogenous induction. CRC cells treated with 3D-Exo-miR-99b-5p displayed significantly reduced proliferation, invasion and migration abilities in comparison to those treated with 3D-Exo-NC (Fig. 3F–I). The findings from the wound healing assay for LoVo and SW480 cell migration were consistent with those of the Transwell assay (Fig. 3J–K). In addition, EMT-related proteins, including E-cadherin, vimentin and N-cadherin, were detected by western blot assay. The results indicated that Vimentin and N-cadherin were downregulated, whereas E-cadherin was upregulated in CRC cells cocultured with 3D-Exo-miR-99b-5p (Fig. 3L–M). IF staining yielded results consistent with those obtained from western blot assay, further confirming the upregulation of E-cadherin and the downregulation of Vimentin (Fig. S1B). Collectively, these findings indicate that exosomes derived from 3D-cultured MSCs efficiently deliver miR-99b-5p to CRC cells, thereby suppressing critical malignant processes such as proliferation, migration, invasion, and EMT.

Fig. 3.

The 3D-Exo-miR-99b-5p derived from MSCs suppressed the growth of CRC cells in vitro. A The morphology of exosomes observed by TEM. B The concentration and particle size distribution of MSCs-exosomes determined by NTA. C Cellular uptake of DiR-labeled exosomes in CRC cells images by confocal microscopy. D–E Real-time PCR analysis of miR-99b-5p level in LoVo and SW480 cells (n = 3). F–K The cell proliferation, migration, and invasion properties of co-cultured LoVo and SW480 cells were detected by using the CCK8, Transwell assays, and wound healing assay (n = 3). L–M The expression of EMT markers (E-cadherin, Vimentin, and N-cadherin) detected by western blot assay (n = 3). Student’s t-test was used to compare two groups, while one-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

The 3D-Exo-miR-99b-5p derived from MSCs promotes ferroptosis and suppresses FGFR3/PI3K/AKt pathway in CRC cells

Treatment of the two CRC cell lines (LoVo and SW480) with the classical ferroptosis inducer Erastin (20 µM, 24 h) effectively initiated ferroptosis cell death. TEM assay revealed morphological features consistent with ferroptosis in both cell types following Erastin exposure (Fig. 4A–B). To confirm the ferroptosis effect of miR-99b-5p in CRC cells, LoVo and SW480 were co-incubation with 3D-Exo-miR-99b-5p. Then, the markers of ferroptosis, including Fe²⁺, GSH, GPX 4 and ROS, were determined by western blot assay, ELISA assay and immunofluorescence (Fig. 4C–D, Fig. S2). Interestingly, the results indicate that after treatment with 3D-Exo-miR-99b-5p, ferroptosis markers such as GSH, and GPX4 decreased, while the Fe²⁺ and ROS levels increased. Furthermore, all these effects were effectively reversed by the ferroptosis-specific inhibitor Fer-1, thereby confirming that miR-99b-5p promotes ferroptosis. This indicates that miR-99b-5p promoted the occurrence of ferroptosis in CRC cells. Additionally, western blot assay indicated a significant downregulation in the expression of FGFR3, phosphorylated AKt (p-AKt), phosphorylated phosphoinositide 3-kinase (p-PI3K) in CRC cells after treatment with 3D-Exo-miR-99b-5p (Fig. 4E–H). To further confirm the downstream of FGFR3, the knockdown or overexpression vectors were transfected into CRC cells (LoVo and SW480). The knockdown or overexpression efficiency of the vectors was shown by real-time PCR and western-blot assay (Fig. 4I–P). After overexpression or knockdown of FGFR3 in CRC cells, the western blot assay was applied to determine the effect of FGFR3 on PI3K/AKt signaling pathway (Fig. 4Q–R). The results show that knockdown of FGFR3 attenuated PI3K/AKt pathway, while overexpression of FGFR3 activated PI3K/AKt pathway in CRC cells. These findings suggest that exosomes derived from 3D-cultured MSCs can efficiently deliver miR-99b-5p to CRC cells, thereby promoting the occurrence of ferroptosis and inhibiting the FGFR3/PI3K/AKt signaling pathway.

Fig. 4.

The 3D-Exo-miR-99b-5p derived from MSCs promotes ferroptosis and suppresses FGFR3/PI3K/AKt pathway in CRC cells. A–B TEM micrographs of LoVo and SW480 cells treated with Erastin and DMSO, red arrow indicated the morphological change of mitochondria. (C) Western blot assay of GPX4 expression in LoVo and SW480 cells. D The levels of Fe²⁺, GSH, ROS and GPX4 in LoVo and SW480 cells detected by ELISA and immunofluorescence (n = 3). E–F The expression of FGFR3 in LoVo and SW480 cells assessed by western blot assay (n = 3); G–H Western blot assay of PI3K, p-PI3K, AKt, and p-AKt expression levels in CRC cells co-cultured with 3D-Exo-miR-99b-5p (n = 3). I–P Verification of overexpression (OE) and knockdown (Si) of FGFR3 in LoVo and SW480 cells through Real-time PCR and western blot assay (n = 3). Q–R Western blot assay of PI3K, p-PI3K, AKt, and p-AKt expression in CRC cells transfected with OE- or Si-FGFR3 vectors (n = 3). Student’s t-test was used to compare two groups, while one-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

The 3D-Exo-miR-99b-5p affects the ferroptosis process of CRC cells by targeting FGFR3

To verify the mechanism of miR-99b-5p in ferroptosis, we focused on the regulatory effect of its downstream target FGFR3. The viability of CRC cells was determined by CCK-8 assay (Fig. 5A–B), and CRC cells induced by Erastin were treated with three cell death inhibitors (Ferrostatin-1, Z-VAD-FMK, Necrostatin-1). The results showed that only Ferrostatin-1 could reverse the ferroptosis process induced by Erastin in CRC cells, while other types of cell death inhibitors failed to restore cell viability, indicating that Erastin only induces ferroptosis in CRC cells rather than other forms of apoptosis. The results of qPCR and Western blot assay (Fig. 5C–E) showed that Erastin treatment could significantly inhibit the mRNA and FGFR3 protein expressions of the two types of CRC cells, while Ferrostatin-1 could reverse this phenomenon. To confirm the role of FGFR3 and 3D-Exo-miR-99b-5p in ferroptosis of CRC cells, Erastin was used to treat CRC cells with overexpression of miR-99b-5p and FGFR3 knockdown/overexpressed to induce ferroptosis, and to determine ferroptosis markers. It was observed that overexpression of FGFR3 could inhibit the increase of Fe²⁺ and ROS levels caused by miR-99b-5p and decrease the GSH and GPX4 levels. The knockdown of FGFR3 enhanced the ferroptosis effect triggered by miR-99b-5p (Fig. 5F–N). In summary, miR-99b-5p directly targets and down-regulates FGFR3, thereby reducing the expression of GPX4 and enhancing the accumulation of ROS, ultimately promoting the ferroptosis process in CRC cells.

Fig. 5.

The 3D-Exo-miR-99b-5p affects the ferroptosis process of CRC cells by targeting and regulating FGFR3. A–B The viability of LoVo and SW480 cells determined by CCK-8 assay (n = 3). C–E FGFR3 expression in LoVo and SW480 cells detected by real-time PCR and western blot assay (n = 3). F–G The levels of Fe²⁺ and GSH in LoVo cells were detected by ELISA (n = 3). I–J The levels of Fe²⁺ and GSH in SW480 cells were detected by ELISA (n = 3). H, K, L The level of ROS in LoVo and SW480 detected by immunofluorescence (n = 3). M–N Western blot assay of GPX4 expression in LoVo and SW480 cells (n = 3). Student’s t-test was used to compare two groups, while one-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

miR-99b-5p regulates the ferroptosis process by inhibiting the PI3K/AKt pathway

To further explore the molecular mechanism by which miR-99b-5p regulates ferroptosis, we focused on its influence on the PI3K/AKt signaling pathway. The Western blot results showed that in the two CRC cells induced by Erastin, the expression of p-PI3K and p-AKt were significantly down-regulated, while no obvious changes were observed in the total proteins of PI3K and AKt. The addition of ferroptosis inhibitor Fer-1 can reverse the above downward trend (Fig. 6A–B). It is suggested that Erastin promotes ferroptosis by inhibiting the PI3K/AKt signaling. Subsequently, to further verify the function of this pathway in miR-99b-5p-induced ferroptosis, we used PI3K activator (740 Y-P) and inhibitors (LY294002) to intervene in the pathway respectively. The markers of ferroptosis were determined by ELISA and Western blot assay. The results showed that after treatment with LY294002, the Fe²⁺ content of LoVo and SW480 cells overexpressing miR-99b-5p significantly increased and the GSH content significantly decreased. However, after treatment with 740 Y-P, the Fe²⁺ levels of the two CRC cells significantly decreased and the GSH content significantly increased (Fig. 6C-D, F–G). ROS fluorescence staining further verified the above results (Fig. 6E, H, I–J). miR-99b-5p significantly enhanced ROS accumulation, LY294002 further amplified this effect, while 740 Y-P significantly weakened the ROS signal. Treatment with LY294002 could further down-regulate the expression of GPX4, while 740 Y-P could effectively reverse the decline of GPX4 induced by miR-99b-5p (Fig. 6K–L). The above results indicate that miR-99b-5p promotes the ferroptosis of CRC cells by inhibiting the PI3K/AKt pathway, reducing the expression of GPX4, and enhancing the accumulation of ROS.

Fig. 6.

The 3D-Exo-miR-99b-5p regulates the ferroptosis process by inhibiting the PI3K/AKt pathway. A–B Western blot analysis of PI3K, p-PI3K, AKt, and p-AKt expression levels in CRC cells after treatment with Erastin or combined with Fer-1 (n = 3). C–J The levels of Fe²⁺, GSH, and ROS in LoVo and SW480 cells detected by ELISA and immunofluorescence (n = 3). K–L Western blot assay of GPX4 expression in LoVo and SW480 cells (n = 3). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Biosafety assessment of iRGD-Exo-miR-99b-5p in vivo

For assessment the biosafety of the engineered exosomes iRGD-Exo-miR-99b-5p, mice were administered iRGD-Exo-miR-99b-5p via tail vein injection, with PBS used as the control. At the end of the observation period, blood samples were collected for serum biochemistry assays, and major organ tissues (heart, liver, spleen, lung, and kidney) of mice were isolated for H&E staining. Additionally, the body weights of mice in each treatment group were measured. No significant difference was found in the body weights between the different groups, which indicates that the engineered exosomes had no effect on the average body weight of mice (Fig. 7A). Serum analysis demonstrated no significant effect on liver function markers (ALK and AST) and kidney function markers (CRE and BUN) from treatment with either PBS or iRGD-Exo-miR-99b-5p (Fig. 7B). H&E staining showed that iRGD-Exo-miR-99b-5p treatment did not induce abnormalities or lesions in any major organ of mice (Fig. 7C), highlighting a more favorable tissue compatibility profile.

Fig. 7.

Biosafety assessment of iRGD-Exo-miR-99b-5p in vivo. A Changes in body weights of the different drug-treated mice during the observation period (n = 3). B Serum biochemistry assays including liver function (ALT and AST) and kidney function (CREA and BUN) (n = 3). C HE stains of major organs collected from mice in each group at the end of the experiment

Targeted anti-tumor therapy of iRGD-Exo-miR-99b-5p in vivo

To assess the tumor targeting ability and biodistribution of the engineered exosomes in vivo, the fluorescence intensities of tumor-bearing DiR-stained exosomes were measured by IVIS at different time points. In order to determine the distribution of the exosomes in the body, main organs and tumors were collected and imaged at 48 h post-injection. IVIS spectrum imaging showed that the fluorescence signals of the tumor in the iRGD-Exo-miR-99b-5p were significantly higher than that in the control group, which indicated that iRGD could observably increase the accumulation of exosomes in the tumor tissues (Fig. 8A–B). Next, we investigated whether iRGD-Exo-miR-99b-5p and 5-FU could act synergistically to inhibit CRC cell viability. Compared with the viability of CRC cells (LoVo and SW480) treated with iRGD-Exo-miR-99b-5p or 5-FU alone, the combined treatment significantly enhanced the proliferation inhibitory effect. The combination index (CI) was used to determine the effect of drug-drug interactions and offered a quantitative definition of the antagonism (CI > 1), synergism (CI < 1), and additive effect (CI = 1), calculated by CompuSyn. Notably, all CI values for the various dose combinations were less than 1 (Fig. 8C–D). These findings suggest a synergistic effect of iRGD-Exo-miR-99b-5p and 5-FU on the inhibition of CRC cell growth. Subsequently, we evaluated whether iRGD-Exo-miR-99b-5p could suppress tumor growth in vivo. The results demonstrated that iRGD-Exo-miR-99b-5p significantly reduced the growth rate, volume, and weight of tumor tissues. Moreover, the combination of miR-99b-5p and 5-FU exhibited enhanced tumor-suppressive effects in vivo (Fig. 8E–G). No significant changes were observed in the average body weight of mice across different treatment groups (Fig. 8H). Tumor tissues from tumor-bearing mice were collected and subjected to H&E and IHC staining for Ki-67 expression. The IHC results revealed a marked decrease in Ki-67 expression in the iRGD-Exo-miR-99b-5p treatment group, with the lowest levels observed in the combined treatment group (Fig. 8I–J). Additionally, we found that Fe²⁺ and ROS levels were elevated, while GSH and GPX4 levels were reduced in tumor-bearing mice treated with iRGD-Exo-miR-99b-5p (Fig. 8K). Western blot assay further confirmed that the protein levels of FGFR3, p-PI3K, and p-AKt were downregulated in tumor tissues from mice treated with iRGD-Exo-miR-99b-5p (Fig. 8L–M). These findings indicate that iRGD-modified 3D exosomes loaded with miR-99b-5p can specifically target tumor sites and significantly inhibit the growth of CRC in vivo. Furthermore, the combination of iRGD-Exo-miR-99b-5p and 5-FU exhibited a more potent anti-tumor effect in xenografted mice. In vivo experimental results also suggest that miR-99b-5p induces ferroptosis by inhibiting the FGFR3/PI3K/AKt pathway, thereby suppressing CRC progression.

Fig. 8.

Targeted anti-tumor therapy of iRGD-Exo-miR-99b-5p in vivo. A Fluorescent images of whole-body imaging of tumor-bearing mice after treatment with DiR-labeled exosomes (n = 3). B Representative images of ex vivo fluorescent signals in the major organs at two days after the last injection (n = 3). C–D Cell viability measured by CCK8 assay after three different treatments in LoVo and SW480 cells (n = 3). Quantitation of synergism and antagonism in iRGD-Exo-miR-99b-5p and 5-FU combination. Fa versus CI plots were generated by CompuSyn. CI > 1, antagonism; CI < 1, synergy; CI = 1, additivity. E–G The tumor weight and volume of mice in each group (n = 5). H Body weight changes in tumor-bearing mice in each group (n = 5). I HE staining and IHC staining of Ki67 in tumor tissues (n = 5). J IHC score for Ki-67 staining in each group (n = 5). K Changes of Fe²⁺, ROS, GSH and GPX expression in tumor tissues of each group (n = 5). L–M Changes of FGFR3, P-PI3K, PI3K, p-AKt, and AKt expression in tumor tissues of each group (n = 3). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between three groups. Data are presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

In this study, exosomes were extracted from 3D-cultured MSCs, and their characteristics were subsequently identified. Our findings indicate that, in comparison to traditional 2D monolayer culture conditions, exosomes derived from 3D cultured MSCs exhibited increased output and they are more easily taken up by CRC cells. In vitro experiments revealed that exosomes derived from 3D cultured MSCs could deliver miR-99b-5p, which possesses anti-cancer properties, thereby inhibiting the proliferation, invasion, and migration of CRC cells. Furthermore, we modified the 3D-exosomes with iRGD and administered them intravenously to tumor-bearing mice. The iRGD-modified exosomes exhibited effective targeting of tumor tissues. Subsequent in vivo experiments demonstrated that iRGD-modified 3D-Exo-miR-99b-5p significantly inhibited CRC growth and enhanced the efficacy of 5-FU in suppressing CRC. The results of both in vitro and in vivo experiments demonstrated that miR-99b-5p induces ferroptosis via the FGFR3/PI3K/AKt signaling pathway, thereby inhibiting the proliferation, invasion, and metastasis of CRC. This study highlights that engineered MSCs-derived 3D exosomes have the potential to function as nanocarriers for the delivery of small molecules with anti-cancer properties, facilitating targeted tumor treatment.

In recent years, preclinical studies have underscored the therapeutic potential of MSCs-derived exosomes [3]. Nonetheless, the translation of exosome-based therapies into clinical practice remains hindered by several significant challenges. Notably, the production of exosomes under routine culture conditions is extremely low, and the large-scale production of clinical-grade exosomes remains a critical technical hurdle that necessitates resolution [12, 13]. To solve this problem, some researchers have explored the use of 3D culture systems to enhance the yield of exosomes secreted by MSCs, and they propose that further investigation into 3D-exosomes can clear the main obstacles for the clinical use of exosomes [6, 7]. Previous studies have demonstrated that 3D culture methods are effective in maintaining cellular pluripotency, enhancing paracrine activity, and increasing exosome production [11]. The 3D culture system effectively simulates the in vivo microenvironment, thereby rendering the cells and exosomes cultured in 3D more representative of their native state [14]. Consistent with these findings, the present study also confirms that the 3D cultivation of MSCs results in an enhanced yield of exosomes and markedly improves the collection efficiency of exosomes.

Several researchers have identified the insufficient targeting capability of stem cell-derived exosomes as a significant barrier to their clinical practice. Engineering modifications of exosomes represent a crucial strategy to improve their tumor-targeting specificity [15, 16]. Sugahara et al. found that iRGD functions as a targeting transmembrane peptide, when co-administered with anti-tumor drugs in tumor-bearing mice, this peptide facilitated the penetration of the drugs into challenging tumor tissues without increasing toxicity to healthy cells, thereby significantly improving drug delivery and anti-tumor efficacy [17]. Multiple studies have corroborated that iRGD-modified engineered exosomes can specifically recognize tumor cells, encapsulate anti-tumor agents, and deliver them to tumor tissues, effectively inhibiting tumor growth [8, 18]. In the present experiment, 3D-exosomes derived from stem cells were engineered modified with iRGD and administered to tumor-bearing mice via the tail vein. The findings indicated that the engineered exosomes successfully targeted tumor tissues, aligning with previous reports. In addition, the safety of iRGD-modified 3D-exosomes was evaluated through in vivo experiments. The results indicated no toxic effects on normal organs, as evidenced by the absence of body weight loss and the maintenance of normal liver and kidney function. These findings suggest that the newly engineered exosomes in this study possess both tumor-targeting ability and a favorable safety profile.

Based on the remarkable tumor-targeting ability of iRGD-modified 3D-exosomes, this study aims to incorporate biologically active molecules with anti-cancer properties into engineered exosomes to further investigate their efficacy in the targeted treatment of CRC. miRNAs are well documented for their ability to be encapsulated with exosomes and subsequently modulate gene expression in recipient cell types [19]. miR-99b-5p has been shown to possess tumor-suppressing functions in various malignant tumors, such as hepatocellular carcinoma [20] and neuroblastoma [21]. Our previous study [10, 22] has established that miR-99b-5p is a miRNA with tumor-suppressor function. Exosomes derived from MSCs have been shown to inhibit the invasion and metastasis of CRC by delivering miR-99b-5p. Building upon these findings, the present study involved the incorporation of miR-99b-5p into iRGD-modified 3D-exosomes. These engineered exosomes exhibit the capability to precisely target tumor tissues and significantly inhibit the proliferation, invasion and metastasis of CRC, as evidenced by both in vivo and in vitro experiments.

The results of this study are consistent with our previous work, further confirming the tumor-suppressive role of miR-99b-5p in CRC. In our prior study [10], we confirmed FGFR3 as a direct target of miR-99b-5p through dual-luciferase reporter assays and observed an inverse correlation between miR-99b-5p and FGFR3 expression in clinical tissue samples. Combined with the inhibitory effects of iRGD-Exo-miR-99b-5p on CRC cell proliferation, migration, and invasion observed in the current study, we hypothesize that its anti-tumor effects may be partially mediated through targeting FGFR3 and related signaling pathways. This provides a molecular basis for understanding the therapeutic mechanism of engineered exosome-delivered miR-99b-5p and enhances the clinical translational potential of this strategy.

Exosome-mediated miRNAs play a crucial role in regulating the expression of target genes, highlighting the necessity of identifying miRNA-mRNA interactions to better understand the regulatory networks governed by miRNAs [23]. Our data demonstrated that in CRC, miR-99b-5p functions as a tumor suppressor by directly targeting fibroblast growth FGFR3, leading to the regulation of the PI3K/AKt signaling pathway. This pathway is a critical intracellular mechanism that governs a wide array of cellular processes, including growth, survival, metabolism, and motility. Its aberrant activation is a characteristic feature of numerous cancers, playing a substantial role in tumorigenesis and cancer progression [24]. Dysregulation of the PI3K/AKt pathway often arises from genetic mutations and alterations in both upstream and downstream signaling components, leading to uncontrolled cellular proliferation and survival. Previous studies support the critical role of the PI3K/AKt pathway in CRC metastasis and highlight the therapeutic potential of targeting this pathway [25]. FGFR3 is a transmembrane receptor tyrosine kinase that plays a pivotal role in regulating essential cellular processes, such as proliferation, differentiation, and survival. Evidence suggests that FGFR3, particularly through its downstream PI3K/AKt pathway, significantly contributes to the regulation of tumor cell invasion and metastatic potential [26]. In this study, we found that MSCs-derived 3D-exosomes overexpressing miR-99b-5p could lead to the inhibition of FGFR3 and suppress the activation of PI3K/AKt signaling pathway, thereby impeding the progression of CRC.

Ferroptosis, a regulated form of cell death characterized by iron-dependent lipid peroxidation, has emerged as a promising target for cancer treatment [27]. Recent studies have underscored the therapeutic potential of MSCs-derived exosomes in modulating ferroptosis. These exosomes can transport bioactive molecules, such as miRNAs, which regulate vital pathways associated with ferroptosis, thereby influencing tumor progression and chemoresistance [28]. For instance, exosomal miR-522 has been shown to inhibit ferroptosis in gastric cancer cells by targeting arachidonate lipoxygenase 15, resulting in reduced lipid peroxide accumulation and increased chemoresistance [29]. The capacity of MSC-derived exosomal miRNAs to modulate ferroptosis is especially pertinent in oncology, as they can either facilitate or impede tumor progression contingent upon the specific miRNAs present and the characteristics of the tumor microenvironment [30]. In the present study, the results indicate that the overexpression of miR-99b-5p can inhibit the FGFR3/PI3K/AKt signaling pathway, and promote cellular ferroptosis, thereby suppressing the proliferation of CRC cells.

Exosomes derived from MSCs have been utilized for the targeted delivery of RNA-based therapeutics in vivo, particularly to tumor tissues, demonstrating significant promise as naturally occurring nanocarriers. In this study, an iRGD-modified, MSCs-derived 3D exosome system was developed and evaluated for its ability to efficiently deliver a therapeutic cargo (miR-99b-5p) to CRC cells, resulting in significant suppression of tumor progression in both cellular and animal models. The study successfully introduced an innovative strategy that employs 3D culture techniques to substantially increase exosome yield, alongside iRGD modification to enhance tumor-specific targeting in vivo, thereby augmenting therapeutic efficacy of exosome-based therapies. Nevertheless, several limitations of the current study warrant discussion. Firstly, although the 3D culture system significantly increased exosome yield, the feasibility of scaling up to meet Good Manufacture Practice (GMP) principles and ensuring consistent quality control for clinical applications remains unresolved. Secondly, while preliminary toxicity assessments in healthy mice showed no adverse effects, these findings are insufficient to confirm long-term safety. Further comprehensive evaluations of systemic toxicity, immunogenicity, and potential off-target effects of both the iRGD-modified exosomes and the delivered miR-99b-5p are necessary. Lastly, the clinical relevance and therapeutic advantages of iRGD-modified 3D exosomes require further validation through large-scale, multicenter clinical trials.

Conclusions

iRGD-modified 3D exosomes derived from MSCs and loaded with miR-99b-5p can specifically target tumor sites, thereby significantly enhancing their antitumor efficacy in CRC. Mechanistically, the exosomal delivery of miR-99b-5p suppressed FGFR3 expression, which consequently inhibited the activation of the PI3K/AKt pathway and promoted ferroptosis, ultimately attenuating CRC progression. These findings suggest that engineered exosomes with appropriate surface modifications can efficiently and precisely deliver antitumor molecules to tumor sites with reduced treatment-related adverse effects. Further investigations in this domain are anticipated to facilitate the clinical translation of engineered exosomes for targeted cancer therapy.

Author contributions

Study concept and design, supervised all experiments: NSF, ZLT and HBL; Completed the experiment: LXH, LFW, NSF, LF and LXL; Performed animal experiments: LXH and LFW; Measuring and evaluating the results of the animal experiments: LF and LXL; Data analysis and interpretation: NSF, ZLT and HBL; Manuscript writing and review: All authors. All authors have read and approved the manuscript in its current state.

Funding

This study was supported by the Guangxi Natural Science Foundation (2025GXNSFAA069580); the National Natural Science Foundation of China (No. 82260580 and 82560520); the Guangxi Medical University Training Program for Distinguished Young Scholars; the Training Program for 1000 Young and Middle-aged Key Teachers in Guangxi.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Animal experiments were approved by the Animal Experiments Ethics Committee of Guangxi Medical University Cancer Hospital. [Project title: The role and mechanism of engineering mesenchymal stem cell-derived exosomes carrying miR-99b-5p in targeted therapy of CRC]. (Approval number: KY-2022-130, Date of approval: February 25th, 2022). The treatment of animals in this study conforms to the ethical standards of experimental animals. The human CRC cells (LoVo and SW480) used in this research were purchased from the Cell Bank, Chinese Academy of Sciences (Shanghai, China). The human bone MSCs were purchased from Haixing Biosciences Co., Ltd in Suzhou, China. According to the vendor, the original source of these cells followed rigorous ethical standards during the collection process, including the acquisition of institutional ethical approval and informed consent from the donors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.