Overcoming acquired immunotherapy resistance in non-small cell lung cancer using ginsenoside Rb1-loaded, peptide-enhanced exosome delivery systems | Journal of Nanobiotechnology

Cell culture

A549 cells used in this experiment were obtained from ATCC (CCL-185, ATCC, USA) and cultured in RPMI 1640 medium (R4130, Sigma, USA) supplemented with 10% FBS (F8318, Sigma, USA) and 1% penicillin–streptomycin (V900929, Sigma, USA) at 37 °C in a 5% CO₂ humidified incubator. The A549 cell line with PI3K E545K mutation was established by Genesray Technology (Genesray Technology, China).

The 344SQ and 344SQ_R cells were cultured at 37 °C in a humidified incubator with 5% CO₂ in RPMI 1640 medium (R4130, Sigma, USA) supplemented with 10% FBS (F8318, Sigma, USA) and 1% penicillin–streptomycin (V900929, Sigma, USA).

Isolation of CSC-Exo

Cancer stem cell (CSC) spheres were enriched from A549 cells via sphere formation assays under serum-free and non-adherent conditions. The enrichment of CSCs was validated by flow cytometry analysis of surface stem cell markers CD44 (14–0441-82, Thermo Fisher, USA) and CD133 (12–1338-42, Thermo Fisher, USA) on A549-CSCs. The culture supernatant of collected CSCs was sequentially centrifuged at 4 °C to remove cells and debris: 300 ×g for 10 min, 2000 ×g for 10 min, and 10,000 ×g for 30 min. The supernatant was resuspended in PBS and filtered through a 0.22 μm sterile filter (Millipore). The filtrate was then ultracentrifuged at 140,000 ×g for 70 min at 4 °C using an Optima L-100 XP ultracentrifuge (Beckman Coulter). The pellet was resuspended in PBS and subjected to a second ultracentrifugation at 140,000 ×g for 70 min to obtain CSC-derived exosomes (CSC-exo).

Synthesis of DSPE-PEG-TMTP1

The TMTP1 peptide (ASN-Val-Val-Arg-Gln-Cys) was synthesized using standard Fmoc solid-phase peptide synthesis [49]. The peptide was then conjugated to DSPE-PEG-COOH via an amidation reaction to form DSPE-PEG-TMTP1 (Figure S1A). Briefly, DSPE-PEG-TMTP1 was synthesized by conjugating TMTP1 with DSPE-PEG-COOH through an amidation reaction. DSPE-PEG-COOH (18 mg) was dissolved in distilled water and catalyzed with EDC (61.2 mg) and NHS (37.2 mg) for half an hour. TMTP1 (7 mg) was added to the reaction mixture and rotated in the dark for 10 h. Unconjugated molecules were removed by overnight dialysis (cut-off molecular weight of 1000 Da) using a YOBIOS YD20DG28 membrane. DSPE-PEG-TMTP1 purity was confirmed by HPLC (Agilent 1200, USA) and compound identity was verified by LC–MS (Agilent 1200 HPLC & 6410 Triple Quad, USA), confirming successful synthesis (Figure S1B-C). The DSPE-PEG-TMTP1 product was freeze-dried and stored as a powder at − 20 °C until further use.

Preparation of TMTP1-Exo

The exosomal lipid bilayer allows for the hydrophobic insertion of lipophilic moieties. Lipid-anchored targeting peptides can be incorporated into the exosome membrane through simple mixing and incubation. DSPE-PEG is a widely used scaffold for anchoring targeting molecules on the exosome surface [50]. DSPE-PEG-TMTP1 and the requisite amount of CSC-exo were co-incubated at 37 °C in the dark for 2 h, followed by centrifugation at 16,000 g for 30 min to obtain TMTP1-exo. Successful conjugation of TMTP1 with CSC-exo was confirmed by labeling TMTP1 with Rhodamine (Rhd) (HY-Y0016, MedChemExpress, USA) to form Rhd-TMTP1 and labeling CSC-exo with PKH67 (HY-D1421, MedChemExpress, USA). After the reaction with TMTP1, co-localization of Rhd-TMTP1 with PKH67-labeled CSC-exo was observed using laser scanning confocal microscopy (Leica, STELLARIS 5, Germany), indicating successful binding of TMTP1 to CSC-exo.

Drug loading and release testing

Using electroporation technology (Nepa Gene, Japan), rb1 (HY-N0039, MedChemExpress, USA) was loaded into T-exo. Subsequently, flow cytometry was employed to detect the fluorescence of FITC-labeled Rb1 within T-exo, confirming the successful encapsulation of Rb1. Rb1@T-exo was dispersed in PBS at a concentration of 1.0 mg/mL, and 2 mL of the prepared solution was placed into a dialysis bag submerged in a 50 mL tube containing 10 mL of pH 7.4 PBS solution, then incubated at 37 °C with agitation at 100 rpm. Samples of 2 mL PBS solution were collected at specific time intervals, their concentrations were measured, and an equivalent volume of fresh PBS solution was immediately replenished to maintain the total volume at 10 mL. The content of ginsenoside Rb1 in the samples was quantified using high-performance liquid chromatography (HPLC). A graph depicting the cumulative drug release percentage against dialysis time was plotted to generate an in vitro release profile.

In vivo pharmacokinetic studies

10 BALB/c mice were allowed free access to food and water, and acclimated for 1 week prior to injection. On the day of the experiment, mice (n = 5 per group) were anesthetized with 2% isoflurane and administered 10 mg/kg of free Rb1 or Rb1@T-exo via tail vein. Blood samples (~ 10 μL) were collected at 2.5, 15, 30, 45, 60, 90 min; 2, 4, 6, 8, 12 h; and daily up to 7 days. Samples were collected in capillary tubes containing 20 μL K2-EDTA (19.447.001, Sarstedt, Germany).

Plasma was separated by centrifugation at 2000 ×g for 10 min at 4 °C and stored at –20 °C. 5 μL of plasma was mixed with 10 μL internal standard (1 μg/mL Ketamine-D4) and 100 μL acetonitrile containing 2% formic acid, followed by centrifugation at 20,000 ×g for 15 min. The supernatant was dried using a CentriVap concentrator (VWR Labconco) and reconstituted in 50 μL of 75% methanol. Rb1 quantification was performed using LC–MS/MS [51].

Characterization of Rb1@T-Exo

Transmission Electron Microscopy (TEM): A drop of 20 µL of Rb1@T-exo was placed on a copper grid and left to stand for 3 min. Excess liquid was removed by gently touching the side with filter paper. Subsequently, 30 μL of pH 6.8 phosphotungstic acid solution (79,690, Merck, USA) was added and left for 5 min at room temperature for negative staining. The sample was then air-dried and observed using a TEM (JEM-1011, JEOL, Tokyo, Japan) operating at an accelerating voltage of 80 kV. Images were captured using the side-mount Camera-Megaview III device (Soft Imaging System, Muenster, Germany).

Nanoparticle Tracking Analysis (NTA): Extracellular vesicles from each group were suspended in PBS and diluted 500 times using Milli-Q water. The diluted extracellular vesicles were injected into the sample chamber of a NanoSight LM10 instrument (Malvern, UK) using a sterile syringe, ensuring the absence of air bubbles until the chamber was full. Videos were analyzed using NanoSight version 2.3 software (Malvern, UK).

Identification of extracellular vesicle surface markers using WB: Extracellular vesicles were resuspended in RIPA lysis buffer (Aspen Biotech, Wuhan, China, AS1004), and surface markers CD81, CD63, Alix, as well as endoplasmic reticulum marker Calnexin, were detected using WB analysis. Refer to the following WB section for antibody information. Each experiment was performed in triplicate.

For drug loading quantification, Rb1@T-exo was dissolved in acetonitrile (Sigma, 34,888) and analyzed using a UV–Vis spectrophotometer (Cary 4000, Agilent Technologies, USA) at 203 nm. Standard curves (R² > 0.999) were constructed using ten concentrations ranging from 5 to 500 µg/mL (each in triplicate). Encapsulation efficiency (EE) and drug loading content were calculated using formulas (1) and (2), respectively. Each sample was tested in triplicate [51].

Uptake of Rb1@T-Exo by tumor cells

A549 cells (5 × 10⁵ cells per well) were seeded onto cell culture slides and allowed to reach 75% confluency. Cy5.5-labeled exo (100 mg/ml), T-exo (100 mg/ml), Rb1@T-exo (100 mg/ml), and an equal volume of PBS were separately co-cultured with A549 cells for 24 h. The cells were stained with FITC-Phalloidin (ab235137, Abcam, UK) and DAPI (C1002, Beyotime, China) for imaging on an Olympus IX81 fluorescence microscope. Before DAPI staining, the slides were washed three times. Images were captured and analyzed using Image J software.

Subcutaneous xenograft model

Healthy immunodeficient nude mice (BALB/c, nu/nu, male, 6–8 weeks old, weighing 18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (211, Beijing, China). The mice were individually housed in SPF-grade animal facilities with a humidity of 60%-65% and temperature maintained at 22–25 °C. They were kept on a 12-h light–dark cycle and provided ad libitum access to food and water. After a one-week acclimatization period, the mice were assessed for their health status before the commencement of the experiments. Our institutional animal ethics committee approved all animal experiments, complied with local regulations on the management and use of laboratory animals, and strictly adhered to international guidelines on animal experimentation ethics to ensure both the welfare of the animals during the experiments and the scientific validity of the study.

PI3K-mutated A549 cells (2 × 10⁶ cells/mouse) were injected into the armpits of the mice. The BALB/c nude mice were randomly divided into 5 groups (n = 6 per group): the Control group, exo group, T-exo group, Rb1@T-exo group, and Rb1 group. On days 7, 14, and 21 post-tumor implantation, PBS, exo, T-exo, and Rb1@T-exo at equivalent volumes were intravenously injected through the tail vein, with concentrations of exo, T-exo, and Rb1@T-exo being 10 mg/kg per injection. The Rb1 group received Rb1 (5 mg/kg) via tail vein injection on days 7, 14, and 21 post-tumor implantation. Tumor volume was measured every four days using the formula: V = (width)² × length/2. On the 28th day, the mice were euthanized, tumors were excised, and their weights were recorded [52,53,54].

Targeting ability of Rb1@T-exo in tumors

To evaluate the tumor-targeting ability of Rb1@T-exo in vivo, exo, T-exo, and Rb1@T-exo were labeled with Cy5.5. In the A549 cell subcutaneous xenograft mouse model, mice were injected with PBS, Cy5.5-labeled exo, T-exo, or Rb1@T-exo via the tail vein. The mice were then dissected to collect tumors, heart, liver, spleen, lungs, and kidneys. The Cy5.5-labeled exo fluorescent images were captured using the IVIS Spectrum Imaging System (PerkinElmer, Waltham, MA, USA).

Bone metastasis model

A plasmid containing the luciferase reporter gene (pGL6-TA, D2105-1 μg, Beyotime, China) was transfected into PI3K E545K mutation A549 cells using Lipo8000™ transfection reagent (C0533-0.5 ml, Beyotime, China). The cells were stably transfected by selection in a medium supplemented with Neomycin sulfate (HY-B0470, MedChemExpress, USA). To investigate bone metastasis, BALB/c nude mice (4–6 weeks old) were anesthetized, and 1 × 10⁵ A549 cells were injected into the left ventricle in a volume of 100 μl. Osteolytic lesions in the bone were observed by micro-computed tomography (μCT). For bioluminescence imaging (BLI) analysis, D-luciferin (200 mg/kg, MedChemExpress, USA) was intraperitoneally injected into the mice, followed by anesthesia with isoflurane 12 min after luciferin injection. BLI images were acquired using the IVIS imaging system (Xenogen, Alameda, California). The results were published in two articles [55, 56].

Acquired resistance to immunotherapy NSCLC model

The 344SQ cell line (344SQ_P) is a metastatic mouse lung cancer cell line generously provided by Dr. Jonathan Kurie (MD Anderson). The construction of the 344SQ_R cells, harboring the PI3K E545K mutation, was completed by Genesray Technologies (Genesray Technologies, China). To generate the anti-PD-1 cell line (Figure S2A), 344SQ_P cells (0.5 × 10⁶ cells, resuspended in 50 μL PBS) were subcutaneously injected into the legs of female 129S2/SvPasCrl mice (129/Sv mice) (12–16 weeks old) from Vital River Laboratories (217, Beijing, China). Subsequently, the mice were intraperitoneally injected with anti-PD-1 (RMP1-14, 10 mg/kg, HY-P99144, MedChemExpress, USA) starting from the 4th-day post-tumor cell injection, twice a week for a total of four injections. Tumors nonresponsive to anti-PD-1 therapy were isolated from the treated mice, digested into single cells, cultured in vitro for approximately 2 to 3 weeks, and subjected to four consecutive in vivo passages in syngeneic mice. These cells resisted anti-PD-1 therapy in vivo, establishing the PI3K E545K mutation anti-PD-1-344SQ_R cell line.

The 344SQ_R cells were cultured in a humidified incubator at 37 °C with 5% CO₂ in RPMI 1640 medium (R4130, Sigma, USA) supplemented with 10% FBS (F8318, Sigma, USA) and 1% penicillin–streptomycin (V900929, Sigma, USA). Regular tests were conducted on the cells to confirm the absence of mycoplasma contamination. Both 344SQ_P and 344SQ_R cell lines were authenticated by DDC Medical (Fairfield, Ohio) using short tandem repeat (STR) DNA fingerprinting technology. The construction of the PI3K E545K mutation 344SQ_R cells was carried out by Genesray Technologies (Genesray Technologies, China) [57]. All animal experiments were approved by our institution’s animal ethics committee and complied with local guidelines for managing and using experimental animals.

The details and protocols for establishing a PD-1/PD-L1 inhibitor-resistant NSCLC mouse model followed previous studies. In brief, for the PD-1-resistant monotherapy tumor model, female 129 Sv/Ev mice aged 8 to 10 weeks were subcutaneously injected with 1 × 10⁵ 344SQ_R cells into the right leg to establish the tumor model [58, 59]. Following the anti-PD-1 treatment, tumors derived from 344SQ_P cells showed significant regression, while anti-PD-1 did not affect the in vivo tumor growth of 344SQ_R cells (Figure S2B). Hematoxylin and eosin (H&E) staining revealed that 344SQ_P tumors retained some degree of glandular formation, a characteristic of the differentiated adenocarcinoma morphotype, whereas 344SQ_R tumors exhibited more diffuse and poorly differentiated characteristics. Furthermore, compared to 344SQ_P tumors, 344SQ_R tumors displayed significantly increased mitotic figures, nuclear pleomorphism, and reduced immune cell infiltration (Figure S2C).

Mice were randomly assigned to four groups (n = 6 per group): Control group, Nivolumab (T9907, TargetMol, USA) group, Rb1@T-exo group, and Rb1@T-exo + Nivolumab group. On days 7, 14, and 21 post-tumor implantation, intravenous injections were administered via the tail vein with PBS, PBS, Rb1@T-exo, and Rb1@T-exo, each time at a concentration of exo, T-exo, and Rb1@T-exo (10 mg/kg). The Nivolumab group and Rb1@T-exo + Nivolumab group received Nivolumab intraperitoneally for four consecutive weeks post-tumor implantation (30 mg/kg, twice weekly).

Cell counting kit-8 (CCK-8) assay

The cell proliferation was assessed using the CCK-8 assay kit (40203ES, Yeasen, Shanghai, China). Cells from different groups were seeded into 96-well plates, and each well was supplemented with 10 μL of CCK-8 solution. After incubation at 37 °C for 2 h, the absorbance value (A) was measured using a Multiskan FC microplate reader (51,119,080, Thermo Fisher Scientific, USA) at a detection wavelength of 450 nm. Three replicate wells were set up for each group, and the average value was calculated. For determining cell viability, the cell proliferation rate was calculated using the formula: Cell Proliferation Rate (%) = (Experimental group absorbance value−Blank control absorbance value)/(Control group absorbance value−Blank control absorbance value) × 100%.

Clonogenic assay

After rinsing the well-growing cells of each group with PBS (P2272, Sigma, USA) twice, trypsin (T2600000, Sigma, USA) digestion was performed, and an appropriate amount of complete culture medium was added to create a single-cell suspension. The cells were counted, and 2 mL of cell suspension (500 cells/mL) was seeded into a six-well plate and gently agitated to ensure uniform distribution. The cells were then cultured at 37 °C in a 5% CO₂ incubator for 7–14 days until visible white colonies appeared at the bottom of the well. After two PBS washes, the cells were fixed with 4% paraformaldehyde (158,127, Sigma, USA) and stained with 0.5% crystal violet staining solution (V5265, Sigma, USA) for 15 min. Colonies containing more than 50 cells were counted under a stereomicroscope.

Transwell assay

The cells were counted in a medium containing 10% FBS, then adjusted to a density of 1 × 10⁵ cells/mL in a medium without FBS. For the invasion assay, Matrigel (354,234, Corning, USA) was coated on the upper chamber membrane of the Transwell. Subsequently, 100 μL of cell suspension was added to the upper chamber of the Transwell, with the lower chamber filled with a complete medium containing 10% FBS as a chemoattractant. After incubating for 24 h at 37 °C and 5% CO₂, non-invading cells on the upper chamber were removed with a cotton swab, while cells that migrated to the lower chamber were stained with 0.1% crystal violet. Five random fields were selected and counted under an inverted microscope. Matrigel coating was not needed in the migration assay, and the remaining steps were the same.

Scratch wound healing assay

Cells from each group, in a good growth state, were prepared as single-cell suspensions using the previously described method. The cells in each group were counted and seeded into a six-well plate with a 2 mL cell suspension (8 × 10⁵ cells/mL). The cells were then cultured in a CO₂ incubator at 37 °C until a growth density of 90–100%. Two parallel straight scratch marks were created on the plate surface using a 200 μL pipette tip, followed by two washes with PBS to remove suspended cells. The cells were then cultured in a medium without FBS. After an additional 24 h of incubation, images were captured, and the scratch closure rate was calculated.

Flow cytometry analysis of cell proliferation and apoptosis

Cells from each group or tumor tissues were collected and subjected to flow cytometry to detect cell proliferation using the CellTrace™ CFSE Cell Proliferation Kit (C34554, ThermoFisher, USA).

Cells from each group or tumor tissues were collected and analyzed for apoptosis using the Annexin V-FITC Apoptosis Detection Kit (C1062M, Beyotime, China) via flow cytometry. The collected cells were stained with Annexin V and PI in 1 × binding buffer at room temperature for 15 min. Subsequently, the stained cells were analyzed using the FACSCelesta flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with FlowJo V10 software (FlowJo, OH, USA), with apoptotic cells located in quadrants Q2 and Q3; Q2 represents mid-stage apoptotic cells, and Q3 represents early apoptotic cells.

Acquisition of high-throughput transcriptome sequencing data

Tumor tissue samples from the Control group (n = 3) and Rb1@T-exo group (n = 3) in an immune therapy-acquired resistance NSCLC model with the PI3K E545K mutation were promptly delivered to the laboratory for sample processing and RNA extraction. Total RNA from each sample was extracted using Trizol reagent (catalog number 16096020, ThermoFisher, New York, USA) following the manufacturer’s instructions. The RNA concentration, purity, and integrity were assessed using the Qubit^®2.0 Fluorometer^® (Q33216, Life Technologies, CA, United States) with the Qubit® RNA Analysis Kit (HKR2106-01, Shanghai Bogoo Biotechnology Co., Ltd.), a Nanodrop spectrophotometer (IMPLEN, California, USA), and the Bioanalyzer 2100 system with the RNA Nano 6000 Analysis Kit (5067–1511, Agilent). Once the total RNA content of each sample met the experiment standards in terms of concentration, purity, and integrity, 3 μg of each sample was utilized as input material for RNA sample preparation. Following the manufacturer’s recommendations, the NEBNext^® UltraTM RNA Library Prep Kit (E7435L, NEB, Beijing) suitable for Illumina^® (Nebraska, USA) was utilized to generate cDNA libraries, and the library quality was assessed using the Agilent Bioanalyzer 2100 system. Subsequently, the indexed samples were clustered using TruSeq PE Cluster Kit v3 cBot HS (Illumina) (PE-401–3001, Illumina) on the cBot cluster generation system, followed by sequencing of the library preparations on the Illumina HiSeq 550 platform post-cluster generation.

Quality control of high-throughput sequencing data

The quality of paired-end reads of the raw sequencing data was assessed using FastQC software v0.11.8. The raw data was processed using Cutadapt software 1.18 to eliminate Illumina sequencing adapters and poly(A) tail sequences. Reads with N content exceeding 5% were filtered using a Perl script. Reads with a base quality of at least 20 covering 70% of the bases were extracted using FASTX Toolkit software 0.0.13. BBMap software was employed to repair the paired-end sequences. Finally, the filtered high-quality read fragments were aligned to the mouse reference genome using hisat2 software (0.7.12).

Transcriptome sequencing data bioinformatics analysis

DEGs between the Control and Rb1@T-exo samples were selected using the Xiantao Academic website (with |log2FC|> 2 and p-value < 0.05 as thresholds), followed by generating volcano plots and heatmaps. Further analyses were performed on the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The DEGs were imported into the STRING database (https://string-db.org/) for protein interaction analysis with a species limit of mice and a minimum required interaction score of 0.4. PPI network analysis was conducted using the “count” package in R, sorting nodes based on Degree values to identify the top 20 highly interconnected genes. Additionally, hub genes were identified within the maximum clique centrality (MCC) algorithm using the cytoHubba plugin.

Flow cytometry phenotypic analysis

Single-cell suspensions of cells from different groups of mouse tumor tissues were prepared for flow cytometry phenotypic analysis. For T cell analysis, staining was performed using antibodies specific for PE-CD8 (12–0081-82, 1:50, ThermoFisher, USA, FITC-PD-1 (PA5-35,010,1:50, ThermoFisher, USA), FITC-CD45 (11–0451-82, 1:50, ThermoFisher, USA), FITC-Ki67 (11–5698-82, 1:50, ThermoFisher, USA), eFluor™ 450-IFN-γ (48–7311-82, 1:50, ThermoFisher, USA), and FITC-Granzyme B (11–8898-82, 1:50, ThermoFisher, USA). Cell proliferation of T cells was analyzed using the CellTrace™ CFSE Cell Proliferation Kit (C34554, ThermoFisher, USA).

For macrophage analysis, staining was conducted with antibodies specific for APC-CD206 (17–2061-82, 1:50, ThermoFisher, USA) and PE-CD86 (12–0862-82, 1:50, ThermoFisher, USA).

Flow cytometry analysis was performed using the FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, California, USA).

ELISA

Cell culture supernatants from each group were collected and analyzed for TNF-α (ab208348, Abcam, UK), IL-10 (ab255729, Abcam, UK), Granzyme B (ab238265, Abcam, UK), and INF-γ (ab100689, Abcam, UK) levels using ELISA kits following the manufacturer’s instructions. Subsequent analysis was performed accordingly.

H&E staining

The tissue slices were deparaffinized in water after sectioning, followed by H&E staining using the staining kit (PT001, Shanghai Bogu Biological Technology Co., Ltd., Shanghai, China) per the manufacturer’s instructions. The main steps for H&E staining were as follows: hematoxylin staining at room temperature for 10 min, followed by rinsing in running water for 30–60 s; differentiation in 1% hydrochloric acid alcohol for 30 s, rinsing in running water and soaking for 5 min; eosin staining at room temperature for 1 min; dehydration in a series of alcohol gradients (concentrations of 70%, 80%, 90%, 95%, 100%), with each gradient dehydrated for 1 min; clearing in xylene for 1 min, two rounds of transparency in xylene I and II, each for 1 min; mounting with neutral gum in a ventilated hood and finally observing the morphological changes of the samples under an optical microscope (BX50; Olympus Corp., Tokyo, Japan) by taking photographs.

For bone metastasis research, the hind limb long bones were extracted, fixed in 10% formalin, decalcified, dehydrated in graded alcohol, embedded in paraffin, and 5 μm thick sections of the decalcified tibia tissue were prepared for H&E staining.

Immunohistochemical staining

The samples were baked at 60 °C for 20 min after embedding and sectioning. Subsequently, the sections were sequentially immersed in xylene solution, with 15 min of soaking in fresh xylene each time, followed by a 5-min immersion in absolute alcohol, a second change of absolute alcohol with another 5-min immersion, and finally rehydration in 95% and 70% alcohol for 10 min each. Each slide was treated with 3% H₂O₂ and soaked at room temperature for 10 min to block endogenous peroxidase. Citrate buffer was added, and the slides were microwave-treated for 3 min, followed by antigen retrieval solution, left at room temperature for 10 min, and washed three times with PBS. The slides were then blocked with normal goat serum blocking solution (E510009, Sangon Biotech (Shanghai) Co., Ltd.) for 20 min at room temperature and subsequently incubated with the diluted primary rabbit Ki67 antibody (ab15580, 1:100, Abcam, Cambridge, UK) overnight at 4 °C. After three PBS washes the next day, the slides were incubated with the secondary goat anti-rabbit IgG antibody (ab6721, 1:1000, Abcam, Cambridge, UK) for 30 min. After PBS washes, the DAB chromogen solution (P0203, Beyotime, Shanghai, China) components A, B, and C were added to the specimens, followed by 6 min of color development, and then counterstained with hematoxylin for 30 s. Subsequent dehydration was carried out with 70%, 80%, 90%, 95% ethanol, and absolute ethanol for 2 min each, followed by two immersions in xylene clearing solution for 5 min each. Finally, the slides were sealed with neutral resin and observed under an upright microscope (BX63, Olympus, Japan). Image analysis software ImageJ was used to measure the staining intensity in each field of view. Each group comprised 6 mice, with 3 slices per mouse and 5 fields of view per slice. The average staining intensity of all fields of view for each mouse was considered the representative value for that particular mouse.

Detection of target gene relative expression by real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from tissues or cells using Trizol reagent (15,596,026, ThermoFisher, USA), and the concentration and purity of the total RNA were assessed at 260/280 nm using NanoDrop LITE (ND-LITE-PR, ThermoFisher, USA). The extracted total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with a gDNA eraser (RR047Q, TaKaRa, Japan). RT-qPCR was performed using the 7500 Fast RT-qPCR system (Part No: 4351106, ThermoFisher, USA). The reaction conditions included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 34 s. The primers for each gene were synthesized by TaKaRa (Table S1), with Gapdh as the reference gene. The relative expression levels of each gene were analyzed using the 2^−ΔΔCt method, where △△Ct = (average Ct value of the target gene in the experimental group—average Ct value of the reference gene in the experimental group)–(average Ct value of the target gene in the control group–average Ct value of the reference gene in the control group). All RT-qPCR experiments were performed in triplicate.

WB

Total protein from tissues was extracted using RIPA lysis buffer containing PMSF (P0013C, Beyotime, China), followed by incubation on ice for 30 min and centrifugation at 8000 g for 10 min at 4 °C to collect the supernatant. The BCA assay kit determined the total protein concentration (ThermoFisher, USA, 23,227). Fifty micrograms of protein were dissolved in 2 × SDS loading buffer, boiled at 100 °C for 5 min, and then subjected to SDS-PAGE gel electrophoresis. The proteins were transferred to a PVDF membrane (88,518, ThermoFisher, USA), blocked with 5% BSA (9048–46-8, Sigma-Aldrich, USA) at room temperature for 1 h, and then incubated overnight at 4 °C with diluted primary antibodies p-PI3K (1:1000, 17,366), p-AKT (1:1000, 13,038), p-mTOR (1:1000, 5536), PI3K (1:1000, 4263), AKT (1:1000, 9272), mTOR (1:1000, 2972) from Cell Signaling Technology, CD63 (ab315108), CD81 (ab109201), Alix (ab275377), Calnexin (ab22595), and β-actin (ab8226) from Abcam. The membrane was then washed with TBST three times for 10 min each, followed by incubation with HRP-conjugated goat anti-rabbit IgG H&L (ab97051, 1:2000) and goat anti-mouse IgG (ab205719, 1:2000) for 1 h at room temperature. After TBST washing, the membrane was placed on a clean glass plate, and an ECL fluorescence detection reagent from abs920 (Absin Bioscience Inc., Shanghai, China) was added. The membrane was photographed using the Bio-Rad imaging system and analyzed with Quantity One v4.6.2 software to determine the relative protein content by calculating the intensity of the protein bands normalized to β-actin. The experiment was repeated three times, and the average values were calculated.

Macrophage and T cell analysis

Bone marrow cells flush from the femur and tibia of C57BL/6 mice (6–8 weeks old) (213, Beijing Vital Harbor Laboratory Animal Co., Ltd., Beijing, China) were seeded in DMEM medium (A4192101, ThermoFisher, USA) containing 10% FBS (F8318, Sigma, USA), 2% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (C0215-100 ml, Beyotime, China), 1% non-essential amino acids (M7145, Sigma, USA), and 1% antibiotic–antimycotic solution (15,240–096, ThermoFisher, USA). To induce differentiation of macrophages, 5 ng/ml of macrophage colony-stimulating factor (M-CSF, 416-ML, R&D Systems) was added every two days for 6 days in culture. Flow cytometry analysis showed a purity of macrophages as CD11b⁺ F4/80⁺ macrophages exceeding 90%. Quantitative PCR (qPCR) and ELISA were performed on CD11b⁺ F4/80 + macrophages using a MoFlo XDP high-speed cell sorter (Beckman Coulter).

Following treatment of 344SQ_R cells with PBS, Nivolumab (0.64 nM), Rb1@T-exo (5 ug/ml), or Rb1@T-exo (5 ug/ml) combined with Nivolumab (0.64 nM) for 24 h, the culture supernatant of each group was collected to incubate macrophages for 48 h before analysis of the harvested macrophages.

CD8⁺ T lymphocytes were purified from the spleens of C57BL/6 mice using the EasySep™ Mouse CD8⁺T Cells Separation Kit (19853_C, Stem Cell Technologies). Isolated CD8⁺ T cells were incubated with CD3-FITC antibody (11–0032-82, 1:50, ThermoFisher, USA) and CD8-APC antibody (47–0081-82, 1:50, ThermoFisher, USA) to confirm the selection efficiency, with flow cytometry confirming a purity of CD8 + T cells > 97%.

The activated CD8⁺ T cells were cultured in RPMI-1640 medium (R4130, Sigma, USA) supplemented with CD3 antibody (2 μg/mL; 11–0032-82, 1:50, ThermoFisher, USA), CD28 antibody (1 μg/mL, 12–0281-82, ThermoFisher, USA), and interleukin 2 (IL-2, 5 ng/mL; 402-ML-100/CF, R&D Systems, USA) for activation.

Following a 24-h co-culture of activated CD8⁺ T cells with 344SQ_R cells at a ratio of 2.5:1, the cells were treated with PBS, Nivolumab (0.64 nM), Rb1@T-exo (5 ug/ml), or a combination of Rb1@T-exo (5 ug/ml) with Nivolumab (0.64 nM) for 24 h. Subsequently, the cells were stained with IFN-γ-eFluor™ 450 (48–7311-82, 1:50, ThermoFisher, USA) and Granzyme B-FITC (11–8898-82, 1:50, ThermoFisher, USA) antibodies for detection of Granzyme B + CD8 + T cells and IFN-γ⁺CD8⁺ T cells using FACSCalibur flow cytometer (BD Biosciences, USA) and analyzed with CellQuest Pro software (BD Biosciences, USA).

Before co-culture, 344SQ_R cells were pretreated for 8 h. Subsequently, T cells were co-cultured with 344SQ_R cells at different effector-to-target cell ratios (E/T ratios) in a 12-well plate for 24 h. Cytotoxic activity of T cells was quantified by measuring lactate dehydrogenase (LDH) concentration and absorbance in the culture supernatant. The percentage of specific cytotoxicity was calculated using the formula: Cytotoxicity = (Experimental LDH release–Spontaneous LDH release) / (Maximum LDH release–Spontaneous LDH release) × 100%. Experimental LDH release is the LDH released during co-culture of effector and target cells, spontaneous release is the LDH released from tumor cells in the absence of effector cells, and maximum LDH release represents the release after adding Triton X-100 to cells (100% LDH release).

Statistical analysis

All data were analyzed using SPSS 22.0 statistical software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 9.5. Descriptive data were presented as mean ± standard deviation (Mean ± SD). An unpaired t-test was used to compare two groups, while a one-way analysis of variance (ANOVA) was applied to multiple group comparisons. The homogeneity of variances was assessed using Levene’s test, and when variances were homogenous, Dunnett’s t and LSD-t tests were used for pairwise comparisons. In cases of inhomogeneous variances, Dunnett’s T3 test was utilized. A significance level of p < 0.05 indicated statistically significant differences between the two groups.