2.1 Tissue samples

During January 2024–November 2024, colorectal cancer (CRC) tissues (n = 20) and nearby healthy tissues (n = 20) were gathered from diseased individuals at the Tumor Hospital of Xinjiang Medical University. The signed informed consent was taken from all patients who had not received any prior therapy. The Ethics Committee of Tumor Hospital of Xinjiang Medical University authorized this study (No. G-2024010). All tissue samples were immediately stored in Liq. N₂ for further experimentation.

2.2 Cell line and treatment

CRC cell lines (LoVo, SW480 and HCT116) had been used for this study [16, 17], and provided by the American Tissue Culture Collection (ATCC, USA). Dulbecco’s Modified Eagle Medium (DMEM; 11965084, Gibco, Rockville, MD, USA) having 10% fetal bovine serum (FBS) was used for cell culturing in humidified incubator with 5% CO₂ at 37 °C. For exosome treatment, exosomes from LoVo cells were co-cultured with HCT116 and SW480 cells. All cell experiments were performed independently in triplicate.

2.3 Exosomes’ isolation and detection

LoVo cells were used to isolate exosomes for culturing in media having exosome-depleted FBS. Briefly, cells and debris were removed by centrifuging cell culture supernatants at 12,000×g for 30 minutes. To pellet exosomes, resulting supernatants were further centrifuged for 2 hours at 120,000×g, followed by washing twice with phosphate buffered saline (PBS). For transmission electron microscope (TEM), the exosomes’ suspension was applied to 200-mesh carbon-coated grids at room temperature for 3 minutes. After drying for one minute, 3% phosphotungstic acid was used to stain grids. Exosomes were visualized using JEM-2000EX TEM (JEOL, Tokyo, Japan) and imaged for analysis.

2.4 Cell transfection

GenePharma (Shanghai, China) provided the pcDNA3.1 vectors targeting TFF3 (TFF3) with negative control (Vector) and shRNAs targeting TFF3 (sh-TFF3) with negative control (sh-NC). LoVo cells underwent transfections employing Lipofectamine 2000 (11668-027, Invitrogen, Carlsbad, CA, USA) as per the company’s instructions. Following transfection, the culture supernatants of diversely administered LoVo cells went through isolation of exosomes for subsequent experiments.

2.5 Western blot

Radio immunoprecipitation assay buffer (RIPA) lysis buffer was used to extract total proteins from tumor tissues or CRC cells. Protein samples were fractionated by 10% ‌sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved to polyvinylidene fluoride (PVDF) membrane (FFP26, Beyotime, Shanghai, China). Membranes after blocking were treated with primary antibodies at 4 °C overnight. Afterwards, incubation was made with secondary antibodies (1/2000; ab7090) for 2 h at room temperature. A chemiluminescence detection kit (89880, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to detect protein expressions.

The primary antibodies: β-actin (1 µg/mL; ab8226; Abcam, Shanghai, China), TFF3 (1/500; ab272927), ‌tumor susceptibility gene (Tsg)101 (1/1000; ab125011), and ‌cluster of differentiation (CD)63 (1/1000; ab134045).

2.6 Immunofluorescence (IF) assay

Red fluorescent membrane dye PKH67 (MIDI67, Sigma-Aldrich, St.Louis, MO, USA) was used to label exosomes by following provider’s manual. After thorough washing, labeled exosomes were isolated by centrifuging at 12,000×g for 30 minutes and further purified using ExoQuick™. DMEM was then used to resuspend labeled exosomes, followed by their addition to HCT116 and SW480 cells for co-culture. After incubation, 10% formaldehyde was applied to fix the cells and 4′,6-Diamidino-2-phenylindole (DAPI) for counterstaining. Fluorescent signals were visualized and imaged using fluorescence microscope (80i, Nikon, Tokyo, Japan).

2.7 CCK-8 assay

SW480 and HCT116 cells (1000 cells/well) seeded into the 96-wells plate and treated with exosomes. Cells viabilities were evaluated by Cell Counting Kit-8 (CCK-8; CK04, Dojindo Laboratories, Kumamoto, Japan). 10 μL CCK-8 solution was added to each well after the treatment, followed by 2 h incubation at 37 °C. A spectrophotometer (ND-ONE-W, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure absorbance at 450 nm.

2.8 Colony formation assay

HCT116 and SW480 cells were cultured in 6-well plates for 14 days to form colonies. 4% paraformaldehyde was used to fix the developed colonies, followed by staining via 0.1% crystal violet. After washing, visible colonies had been counted and analyzed.

2.9 Wound healing assay

HCT116 and SW480 cells were cultured in 6-wells plates till they attained 80–90% confluence. A linear scratch was created by sterile pipette tip (10 μL) across cell monolayer. The wound area was explored regarding cell migration and imaged at 0 and 24 h through light microscope.

2.10 Transwell assay for cell invasion

Matrigel (356234, BD Biosciences, Franklin Lakes, NJ, USA) was used to pre-coat transwell chambers having 8 μm pore membranes (pore size, 8 μM; 3470, Corning, NY, USA). The upper chambers were added with HCT116 and SW480 cells suspended in 200 μL serum-free medium, while lower chambers with 600 μL medium having 20% FBS as chemoattractant. After incubating for 48 h, 4% paraformaldehyde was used to fix invasive cells on lower membrane surface, followed by staining via 0.1% crystal violet. Number of invaded cells was microscopically counted (E200, Nikon, Tokyo, Japan).

2.11 RT-qPCR

TRIzol reagent (15596018, Invitrogen, Carlsbad, CA, USA) was employed to extract total RNA by following company’s manual. PrimeScript™ RT Master Mix kit (RR036A Takara, Dalian, China) was applied to perform reverse transcription of RNA into cDNA. qPCR was carried out by employing SYBR Premix Ex Taq™ kit. The 2^−ΔΔCt methodology was adopted to calculate relative LINC00941 expression level. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control.

LINC00941:

Forward, 5′-CTTCTCTGAACTGCGGCTCA-3′;

Reverse, 5′-GGCCTCCTTGCTGACTGATT-3′.

GAPDH:

Forward, 5′-CTGGGCTACACTGAGCACC-3′;

Reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′.

2.12 In vivo assay

The Ethics Committee of Tumor Hospital of Xin Jiang Medical University approved animal experimentation which was executed in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines (No. IACUC-20220306-27). The Vital River company (Beijing, China) provided the male BALB/c nude mice (n = 16). In alignment with the 4R principles of animal research—reduction, refinement, replacement, and responsibility—10 mice were ultimately used for this study. Mice had been randomly grouped into two and administered by either sh-TFF3-Exo or sh-NC-Exo. After 35 days, 2% pentobarbital sodium was used to euthanize mice for dislocating cervix. Tumors had been harvested and processed for subsequent analysis (Approval No. IACUC-20220306-27).

2.13 IHC assay

4 μm thick sections of tumor tissue embedded in paraffin had been dewaxed, rehydrated, and subjected to antigen retrieval. After blocking, the sections underwent overnight incubation at 4 °C with 1 µg/mL primary Ki67 antibody (ab15580, Abcam, Shanghai, China). Next day, the sections underwent incubation at room temperature with secondary antibody (1:1000; ab7090). Staining was performed by diaminobenzidine (DAB) and counterstaining by hematoxylin. Ki-67 expression was microscopically observed and evaluated (E200, Nikon, Tokyo, Japan).

2.14 Statistical analyses

The data were shown as mean ± standard deviation (SD). GraphPad Prism Software 9 (GraphPad Software, San Diego, CA, USA) was used to perform statistical analysis. Two groups were compared and analyzed by t-test, while multiple groups by one-way analysis of variance (ANOVA). p-value of less than 0.05 had been of statistical significance.

3.1 TFF3 is highly expressed in CRC tissue

Analysis using ̵‌gene expression profiling interactive analysis‌ (GEPIA) online database revealed that TFF3 expression was substantially higher in colon adenocarcinoma (COAD) tissues in comparison to normal (Fig. 1A). Consistently, western blot analysis further confirmed that TFF3 protein levels were upregulated in CRC tissues in comparison to the matched normal (Fig. 1B). These outcomes indicate that TFF3 is overexpressed in CRC.

Fig. 1.Overexpressed TFF3 in CRC tissue. (A) GEPIA online database was used to analyze TFF3 expression in normal and colon adenocarcinoma (COAD) tissues. *p < 0.05. (B) Western blotting was employed to examine TFF3 protein levels in normal (n = 20) and CRC tissues (n = 20). TFF: Trefoil factor; TPM: Transcripts per million.

3.2 TFF3 is upregulated in CRC-derived exosomes

TEM confirmed the isolated exosomes by revealing typical vesicle morphology (Fig. 2A). Western blotting depicted higher expressions of exosomal markers Tsg101 and CD63 in isolated exosomes (Fig. 2B). Notably, CRC-derived exosomes had substantially enhanced TFF3 protein expressions (Fig. 2C). These results suggest enriched TFF3 in CRC cells derived exosomes.

Fig. 2.TFF3 is upregulated in CRC-derived exosomes. (A) Isolated exosomes’ morphology was confirmed by TEM. Arrows indicated exosomes. Scale bar = 200 nm. (B) Protein expressions of Tsg101 and CD63 exosomal markers were detected by western blotting (n = 3). (C) TFF3 protein expression in CRC-derived exosomes was evaluated through western blotting (n = 3). **p < 0.01. TFF: Trefoil factor; CD: Cluster of differentiation; Sup: Supernatant; Exo: Exosomes; CM: Conditioned medium; Tsg: Tumor susceptibility gene.

3.3 Exosomal TFF3 promotes CRC cell growth

IF analysis showed that the PKH-26 fluorescence intensity decreased following TFF3 knockdown and increased with TFF3 overexpression, indicating altered exosome uptake (Fig. 3A). Western blot analysis confirmed that TFF3 protein levels were reduced upon silencing and elevated upon overexpression (Fig. 3B). CCK-8 assays exhibited substantially reduced cell viability with TFF3 knockdown, while it increased with TFF3 overexpression (Fig. 3C). Similarly, colony formation assays demonstrated reduced cell proliferation in the TFF3-silenced group and enhanced proliferation in the overexpression group (Fig. 3D). In conclusion, these outcomes reflected that exosomal TFF3 triggered CRC cell growth.

Fig. 3.Exosomal TFF3 promotes CRC cell growth. Groups had been segregated to Exo, sh-NC-Exo, sh-TFF3-Exo, Vector-Exo and TFF3-Exo group. (A) The fluorescence intensity of PKH-26 was examined through IF assay and analyzed through Image J software. Bar = 20 μm. (n = 3). (B) Western blotting assessed TFF3 protein expression. (n = 3). (C) CCK-8 assays evaluated cells viabilities. (n = 3). (D) Colony formation assay explored cells proliferation. N = 3. **p < 0.01 vs. sh-NC-Exo group; #p < 0.05, ##p < 0.01 vs. Vector-Exo group. TFF: Trefoil factor; Exo: Exosomes; sh-NC: shRNA-negative control; DAPI: 4′,6-Diamidino-2-PhenylIndole‌.

3.4 Exosomal TFF3 enhances cells invasion and migration in CRC

Assays from wound healing depicted that CRC cells’ migration rate was substantially decreased upon TFF3 knockdown and enhanced following TFF3 overexpression (Fig. 4A). Similarly, Transwell assays showed that cell invasion was suppressed by TFF3 silencing and promoted by TFF3 overexpression (Fig. 4B). These results demonstrate that exosomal TFF3 enhances CRC cells’ invasion and migration potential.

Fig. 4.Exosomal TFF3 enhances cells invasion and migration in CRCs. Groups had been segregated to Exo, sh-NC-Exo, sh-TFF3-Exo, Vector-Exo and TFF3-Exo groups. (A) The cell migration was evaluated by wound healing assay. (n = 3). (B) Transwell assay was used to assess cells invasion. (n = 3). **p < 0.01 vs. sh-NC-Exo group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Vector-Exo group. TFF: Trefoil factor; Exo: Exosomes; sh-NC: shRNA-negative control.

3.5 Exosomal TFF3 upregulates LINC00941 to promote CRC progression

RT-qPCR analyses showed substantially downregulated LINC00941 expression following TFF3 knockdown and upregulated upon TFF3 overexpression (Fig. 5A). In addition, cells viabilities were decreased following TFF3 knockdown, however the change was rescued by LINC00941 overexpression (Fig. 5B). Similarly, Transwell and wound healing assays revealed that TFF3 knockdown suppressed cells invasion and migration, while LINC00941 overexpression reversed these effects (Fig. 5C,D). These findings suggest that exosomal TFF3 promotes CRC progression by upregulating LINC00941.

Fig. 5.Exosomal TFF3 upregulates LINC00941 to promote CRC progression. (A) The LINC00941 expression was examined through RT-qPCR. (n = 3). Groups had been segregated to Exo, sh-NC-Exo, sh-TFF3-Exo, Vector-Exo and TFF3-Exo group. **p < 0.01 vs. the sh-NC-Exo group; ##p < 0.01 vs. the Vector-Exo group. (B) Cells viabilities were evaluated by CCK-8 assays. (n = 3). (C) The cells migration by wound healing assay. (n = 3). (D) The cell invasion was examined through Transwell assay. Groups had been segregated to sh-NC + Vector-Exo, sh-TFF3 + Vector-Exo and sh-TFF3 + LINC00941-Exo group. N = 3. **p < 0.01 vs. the sh-NC + Vector-Exo group; ##p < 0.01 vs. the sh-TFF3 + Vector-Exo group. TFF: Trefoil factor; Exo: Exosomes; sh-NC: shRNA-negative control.

3.6 Exosomal TFF3 promotes tumor growth in vivo

Finally, in vivo experiments demonstrated that tumors weight, volume, and size had been significantly reduced following TFF3 knockdown (Fig. 6A). Western blot analysis confirmed a decrease in TFF3 protein expression in tumor tissues from the TFF3-silenced group (Fig. 6B). Consistently, RT-qPCR analysis showed that LINC00941 expression was also downregulated after TFF3 suppression (Fig. 6C). IHC staining revealed a marked reduction in Ki67 expression, indicating decreased cell proliferation in the TFF3 knockdown group (Fig. 6D). These findings indicate that exosomal TFF3 promotes tumor growth in vivo.

Fig. 6.Exosomal TFF3 promotes tumor growth in vivo. Groups had been segregated to sh-TFF3-Exo and sh-NC-Exo group. (A) Tumor growth in mice was evaluated. (n = 8). (B) TFF3 protein expressions were evaluated by western blotting. (n = 8). (C) LINC00941 expression had been confirmed via RT-qPCR. (n = 8). (D) The Ki67 expression was determined through IHC assay. Scale Bar = 200 μm. (n = 8). **p < 0.01. TFF: Trefoil factor; Exo: Exosomes; sh-NC: shRNA-negative control.