Cell culture

HCT116 human colon cancer cell, CT26 mouse colon cancer, MC38 mouse colon cancer cell, and human pancreatic cancer cell were preserved in our laboratory (Hubei Province Key Laboratory of Molecular Imaging, China). The cells were cultured in an RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and incubated at 37 °C in a humidified atmosphere with a 5% CO2 concentration.

Isolation of TMPs

The CT26 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (without extracellular vesicles) after being exposed to ultraviolet irradiation (300 J/m2) for 1 h. The supernatants were used to isolate TMPs after 24 h. Briefly, the supernatants were centrifuged for 60 min at 14,000 g to pellet the TMPs after being centrifuged for 30 min at 3000 g to remove cells. The TMPs pellets were resuspended in a culture medium after being cleaned three times. The TMPs were put through a series of 0.45-m filters, quantified by surface proteins using a BCA Protein Assay Kit (Beyotime, Shanghai, China), and then stored at 80 °C.

Synthesis of N3-TMPs@NAP

The protocol was referred to previous study [34]. Dimethylsulfoxide (DMSO, Solarbio, China) was used as a permeability enhancer by increasing the solubility of NAP and its permeability across the lipid membrane of TMPs. A 4% (v/v) DMSO solution in PBS was used for the NAP encapsulation. TMPs@NAP was fabricated by mixing the TMPs with NAP and incubating them for 12 h. Then 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido (polyethyleneglycol)-2000] (DSPE-PEG-N3, 1 μM) was incubated with TMPs@NAP (TMPs, 1 mg) for 30 min at 37 °C to form N3-TMPs@NAP. The samples were then passed through centrifugal filter devices (100 kDa molecular weight, Amicon®Ultra-15) before further use.

Characterization of N



N3-TMPs@NAP was examined using TEM (Hitachi, Japan), DLS (Malvern Instruments Ltd., Worcestershire, UK) and NTA (PARTICLE METRIX, German). To test the in vitro stability of N3-TMPs@NAP, the hydrodynamic diameters were monitored for 7 days using DLS. The amount of NAP in N3-TMPs@NAP was measured using HPLC. Briefly, NAP was dissolved in DMSO and subsequently diluted to different concentrations with PBS (0, 0.05, 0.1, 0.2, 0.3, and 0.5 μM). The UV peaks, which corresponded to the different concentrations, were measured using HPLC, and the standard curves of NAP were plotted. N3-TMPs@NAP was placed in a dialysis bag (MW: 12,000 Da) and incubated with solution of different PH (7.0, 6.5, 5.0) to quantitatively determine the release profile of NAP, which was detected at 0, 24, 48, and 72 h.

Synthesis of 68 Ga‑L‑NETA‑DBCO

The protocol was referred to previous study [30, 35]. By using HCl (0.05 M) as the eluent and a 68Ge/68 Ga generator, 68GaCl3 was produced. To adjust the pH of a 500-μL 68GaCl3 (187 MBq) solution to 3.7, sodium acetate was added. L-NETA-DBCO (5 nmol) was used to chelate the radionuclide 68 Ga for 10 min at 100 °C. After cooling the mixture, a C18 column was used to purify 68 Ga-L-NETA-DBCO. PET/CT imaging was used to detect the in vivo click reaction that occurred during the conjugation of 68 Ga-L-NETA-DBCO with N3-TMPs@NAP.

In vitro tumor cell binding

The ability of in-vitro tumor cell binding was detected using flow cytometric analysis and fluorescence imaging. N3-TMPs@NAP were incubated with Cy5-NHS for 30 min at 37 °C to form Cy5/N3-TMPs@NAP. Cy5/N3-TMPs@NAP (30 μg/mL) were incubated with CT26 cells at 37℃ for different time points (0 h, 1 h, 6 h, 12 h, 24 h). Flow cytometric was used to detect the fluorescence signals. Cy5/N3-TMPs@NAP (30 μg/mL) or Cy5 (30 μg/mL) were then incubated with CT26 cells at 37 ℃ for 24 h. The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were fixed with paraformaldehyde and observed using a fluorescence microscope (Olympus, Japan). In addition, CT26 cells, MC38 mouse colon cancer cells, and Panc01 human pancreatic cancer cells were incubated with Cy5/N3-TMPs@NAP (30 μg/mL) at 37 °C for 24 h to further studying the capability of homologous targeting of TMPs.

Cell counting kit-8 (CCK-8) assays and live/dead cell staining

CT26 cells were plated in 96-well plates (4000 cells/well) in triplicates and incubated at 37 °C overnight. The cells were subsequently treated with NAP (0.1, 0.5, 1, 2, and 10 μM), vehicle control (0.1% DMSO), or N3-TMPs@NAP (NAP: 0.1, 0.5, 1, 2, and 10 μM) for 24 h. Finally, the cell growth was measured using CCK-8 (Dojindo, Japan), following the manufacturer’s instructions. Briefly, after 1-h incubation with CCK-8 at 37 °C, their OD values (at 450 nm) were detected to calculate cell viability. TMPs were also incubated with CT26 cells at different concentrations (0, 1, 5, 10, 20, 50, and 100 μg/mL) for 24 h. To further detected the in-vitro anti-tumor effect, we used the LIVE/DEAD cell double staining kit (BJBALB, China) according to the manufacturer’s instructions.

Colony formation assay

The colony formation assay was used to assess tumor cell proliferation. CT26 colon cancer cells were plated into 6-well plates (500 cells per well) and treated with control (0.1% DMSO), NAP (0.05 μM), TMPs (1 μg/mL), or N3-TMPs@NAP (NAP 0.05 μM). After 12 days, the cells were fixed in methanol for 15 min, stained with 1% Crystal Violet Staining Solution for another 20 min, and washed 3 times with PBS. The number of colonies was counted. All assays were performed in triplicates.

Cell invasion assay

Utilizing transwell chambers with 8-μm pore size, cell invasion capacity was evaluated. CT26 cells were treated with control (0.1% DMSO), NAP (0.1 μM), TMPs (1 μg/mL), or N3-TMPs@NAP (NAP 0.1 μM) and injected into the Matrigel-coated invasion upper chamber of the inserts, and DMEM 30% FBS was placed in the lower chambers and incubated for 18 h at 37 °C and 5% CO2. After incubation, the cells were fixed in methanol for 20 min and then stained with Crystal Violet stain solution (#C0121, Beyotime). Five fields per well of invasion cells were examined under a microscope. All experimental assays were performed in triplicates.

EdU assay

EdU cell proliferation staining was performed using an EdU kit (BeyoClickTM, EDU-488, China). Briefly, the CT26 cells (2 × 104 cells/well) were seeded in 12-well plates and were incubated at 37 °C overnight. The CT26 cells were treated with control (0.1% DMSO), NAP (0.1 μM), TMPs (1 μg/mL), or N3-TMPs@NAP (NAP 0.1 μM). Subsequently, the cells were incubated with EdU for 2 h, fixed with 4% paraformaldehyde for 15 min, and then permeated with 0.3% Triton X-100 for another 15 min. The cells were then incubated with the Click Reaction Mixture for 30 min at room temperature in a dark place and incubated with Hoechst 33342 for 10 min.

Tumor-bearing mouse models

The mouse experiments were approved by the Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China. The right upper limb of BALB/C mice (female, 6 weeks old, Beijing HFK Bioscience Co., Ltd, China) received a subcutaneous injection of CT26 cells (1 × 106) suspended in 100-μL PBS. The mice were ready for experimentation once the tumor size had reached about 5 mm. Liver metastases in the colon cancer models were also prepared as follows. The spleens of 6-week-old BALB/C mice were exposed using laparotomy. Then, the CT26 cells (5 × 106) suspended in 50-μL PBS were injected into their spleens, returned to the abdominal cavity, and sutured the wounds. When the signs of abdominal distension or locomotive deficit appeared or a tumor was detected by palpation, the mice were killed, and their livers and spleens were harvested.

In vivo animal PET/CT imaging and biodistribution analysis

N3-TMPs@NAP (200 μg) was infused into the CT26 tumor-bearing mice (n = 3 per group) at the various pre-targeted time points using different delivery methods (tail intravenous or oral administration). 68 Ga-L-NETA-DBCO (3.7 MBq) was injected into the mice via their tail veins. The mice were anesthetized with 2% isoflurane, and micro-PET/CT static imaging was performed after 2 h of the injection of 68 Ga-L-NETA-DBCO. The static PET/CT images were collected for 10 min using a small-animal PET/CT scanner (Novel Medical, Beijing, China). The mice were sacrificed after PET/CT imaging (n = 3). Their tissues, including brain, heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, muscle, bone, and tumor tissues, were excised, weighed, and analyzed using a γ-counter. Radioactivity in the organs and tissues was calculated as the percentage of injected dose per gram of tissue (% ID/g) and corrected for radioactive decay. N3-TMPs@NAP was incubated with Cy5-NHS for 30 min at 37 °C to form Cy5/N3-TMPs@NAP. Cy5/N3-TMPs@NAP (100 μg) was infused into the CT26 tumor-bearing mice using different methods (tail intravenous or oral administration). Fluorescence imaging was performed to assess the accumulation of Cy5/N3-TMPs@NAP of tumor tissues.

Assessment of the in vivo antitumor effect

The CT26 tumor-bearing mice were randomly divided into four groups (n = 5 in each group), which were respectively treated with NS (0.1% DMSO), NAP (20 mg/kg), TMPs (10 mg/kg), and N3-TMPs@NAP (NAP 20 mg/kg). After treatments, the tumor sizes and mice body weights were measured every 2 days. On the 14th day, all the mice were sacrificed and their tumor tissues were collected, weighed, photographed, and stored for further histological examinations. Immunohistochemistry was performed to assess the expression levels of proteins Ki67 and CD44 in the tumor tissues. Serums of the NAP and N3-TMPs@NAP group was collected for In-vivo pharmacokinetic parameters. Briefly, serums were precipitated with acetonitrile at different time points (1, 2, 6, 12, 24, and 48 h) after drug injection, and the supernatant obtained was detected by HPLC.

In vivo toxicity studies

After treatments, the mice’s blood and major organs (liver, spleen, kidneys, heart, and lungs) were also collected. The function indicators of liver and kidney, such as ALT, AST, ALP, BUN, and CRE, were measured using a blood biochemical autoanalyzer (Chemray 240, Rayto Life and Analytical Sciences Co., Ltd, China). The major organs (hearts, livers, spleens, lungs, and kidneys) were stained with H&E and examined under an optical microscope (IX73, Olympus, Japan).

Transcriptome sequencing

The CT26 cells were treated with NS (0.1% DMSO), NAP (0.2 μM), PBS, TMPs (30 μg/mL). Total RNA was extracted using TRIzol reagent (#15596026, Invitrogen), and transcriptome sequencing was performed by NOVOGENE (Beijing, China) based on the Illumina platform. The prepared libraries were sequenced on an Illumina NovaSeq platform, and 150-bp paired-end reads were generated.

Western blot analysis

The CT26 and HCT116 cells were harvested and lysed with lysis buffer, containing the phosphatase inhibitors and 1% protease, on ice for 30 min. Then, the cell lysates were centrifuged at 12,000 g for 15 min at 4 °C and the supernatants were collected. The protein concentrations were determined using a protein quantification kit (#P0012S, Beyotime) to ensure that equal amounts of total proteins were loaded into each well of SDS-PAGE gels. The gels were transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated with the primary antibodies overnight at 4 °C. On the second day, the membranes were washed with 1 × TBST for 30 min and incubated with the respective secondary antibodies for 1 h. After incubation, the membranes were washed 3 times with PBS and exposed to X-ray films using ECL detection reagents (#WP20005, Thermo Fisher). The antibodies used in this experiment are as follows; STAT1 antibody (#10144-2-AP, 1:2000), STAT2 antibody (#66485–1-Ig, 1:4000), STAT3 antibody (#10253-2-AP, 1:2000), CD44 antibody (#15675-1-AP, 1:2000), BMI1 antibody (#10832-1-AP, 1:2000), TBK1 antibody(#28397-1-AP, 1:2500) and IRF3 antibody(#11312-1-AP,1:5000) were purchased from Proteintech. GAPDH antibody (#ab8245, 1:3000) was purchased from Abcam. p-TBK1(Ser172) antibody (#5483,1:1000), p-IRF3(Ser386) (#37829, 1:1000) were purchased from Cell Signaling Technology.

Quantitative RT-qPCR assay

Total RNA was extracted using a Trizol reagent (#15596026, Invitrogen). The extracted RNA samples were reverse-transcribed using a PrimeScriptTM RT reagent Kit (#RR047A, TAKARA, JPN). Quantitative real-time PCR was performed using a TB GreenTM Fast qPCR Mix kit (#RR430A, TAKARA, JPN). GAPDH served as the reference gene, and the 2−ΔΔCT method was used to quantify the fold change in gene expression. The primer sequences for RT-qPCR are provided in Additional file 1: Table S1.

RNA interference

Gene-specific siRNA were purchased from Sigma-Aldrich. Tumor cells were transfected with siControl or siRNA in Lipofectamine 2000 (#11668019, Thermo Fisher). 12 h after transfection, replace the transfection medium with DMEM containing 10% FBS. siRNA sequences were shown in Additional file 1 (Table S2).

Chromatin immunoprecipitation (ChIP) and ChIP-qPCR

ChIP was performed using the Chromatin Extraction Kit (#ab117152, Abcam) and ChIP Kit Magnetic-One Step (#ab156907, Abcam), following the manufacturer’s instructions. Purified DNA was analyzed using real-time PCR and TB GreenTM Fast qPCR Mix kit (#RR430A, TAKARA, JPN) according to the manufacturer’s protocol. Primers used for ChIP-qPCR were shown in Additional file 1: (Table S3).


Tumor tissues were fixed with 4% paraformaldehyde, and then dehydrated and embedded in paraffin. From the rehydrated tissue slices, antigen retrieval was performed using the heat-induced antigen retrieval in citrate buffer (Vector Laboratories, CA). The tissues were made permeable with PBS, containing 0.2% TritonX-100, and then treated with 3% hydrogen peroxide to inactivate the endogenous peroxidase. The tissues were washed with PBS, containing 0.05% Tween-20, and then incubated for 2 h with a blocking solution (MOM blocking buffer, Vector Laboratories, CA). The samples were then incubated with CD44 antibody overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Signals were amplified using an ABC kit (Vector Laboratories) and visualized using a 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues were further stained with H&E, dehydrated, and mounted (H5000, Vector Laboratories).

In vivo immune response

Immunofluorescence staining was performed to assess the presence of tumor-infiltrating T cells in the tumor tissues. After treatments, the tumor tissues were collected and incubated with the corresponding antibodies, including CD3 (#17617-1-AP, Proteintech), CD8 (#67786-1-Ig, Proteintech), and CD4 (#66868-1-Ig, Proteintech).

Bioinformatic analyses

Bioinformatics analyses were performed using the R Bioconductor. DESeq2 package was used for differential analysis and obtaining the average value of different genes. The ggplot2 and pheatmap packages were used to draw volcano maps and heat maps. ClusterProfiler package was used for the gene set enrichment analysis (GSEA). GOplot package was used to draw the Gocircle plot. The protein–protein interaction (PPI) network was constructed using the STRING database (https://cn.string-db.org/). Cytoscape software was employed to visualize the interaction network, which was analyzed using the Mcode algorithm to calculate the interconnected subgraphs of a complex PPI network. The gene expression and clinical data of colon adenocarcinoma were downloaded from the TCGA biolinks packages (version 2.14.1). Ggstatsplot package was used to analyze the correlations between different genes in colon cancer. UCSC Genome Browser was used to visualize the STAT1 ChIP-seq signal profiles in the CD44 gene region. The CD44 promoter sequences and putative STAT1-binding sites were obtained from the Eukaryotic Promoter Database (EPD) (https://epd.epfl.ch//index.php) and (http://jaspar.genereg.net).

Statistical analyses

Unpaired or paired Student’s t-tests were used for group comparisons in statistical analyses, and one-way or two-way ANOVA was used for multiple comparisons. GraphPad Prism 8 was used to evaluate statistical significance (GraphPad Software, Inc.). Statistics were considered significant at P0.05. Every value was expressed in terms of means ± standards.