Downloading and analyzing gene expression profiles and clinical data from the TCGA database
The Cancer Genome Atlas-prostate adenocarcinoma (TCGA-PRAD) dataset was retrieved from the TCGA database (https://cancergenome.nih.gov/). The dataset comprises gene expression data and clinical information of patients, such as pathological N staging, T staging, M staging, and Gleason score. The TCGA-PRAD dataset includes 501 tumor samples and 52 normal samples. Pathological N staging consists of 52 normal cases, 348 cases without lymph node metastasis (N0), and 80 cases with lymph node metastasis (N1). Clinical T staging includes 52 normal cases, 177 cases of T1, 175 cases of T2, 53 cases of T3, and 2 cases of T4. Clinical M staging consists of 52 normal cases, 457 cases with no distant metastasis (M0), and 3 cases with distant metastasis (M1). Gleason score distribution includes 52 normal cases, 46 cases with a score of 6, 248 cases with a score of 7, 65 cases with a score of 8, 138 cases with a score of 9, and 4 cases with a score of 10. First-line treatment outcomes include 341 cases of complete remission, 40 cases of partial remission (PR), 30 cases of stable disease (SD), and 29 cases of disease progression (PD). Due to the public nature of the data from the database, no ethical approval or informed consent was required.
Survival analysis: The survival time and status of patients from the TCGA-PRAD database were integrated with gene expression data, and survival analysis was performed using the “survival” package in R (version 4.2.1). Patients were divided into two groups based on the median gene expression levels, and survival analysis was conducted.
ROC curve analysis: ROC analysis of the data was performed using the pROC package.
Box plot analysis: A comparison of the distribution of AURKA expression levels across different clinical stages (T staging, N staging, M staging) and Gleason scores was conducted. The Kruskal–Wallis test was employed to analyze group differences based on the normality of the data distribution, with appropriate parametric or non-parametric statistical tests selected accordingly. Tukey’s multiple comparison test was applied to groups showing significant differences.
All graphs were generated using the “ggplot2” package to visually display the relationship between AURKA expression and clinical parameters. The significance level for all statistical tests was set at 0.05, and significance levels were marked with asterisks in the graphs (*P < 0.05, **P < 0.01, ***P < 0.001).
Analysis of scRNA-seq data
We downloaded tumor tissues from three PCa patients with good prognosis (GSM5793828, GSM5793829, GSM5793831) from the GEO database (http://www.ncbi.nlm.nih.gov/geo/) dataset GSE193337 and tumor tissues from three CRPC patients (GSM6267383, GSM6267384, GSM6267385) from dataset GSE206962.
The data was analyzed using the ‘Seurat’ package in R software. Data quality control was performed using the criteria of 200 < nFeature_RNA < 5000 & percent.mt < 10, and highly variable genes with the top 2000 variances were selected for further analysis.
To reduce the dimensionality of the scRNA-Seq dataset, Principal Component Analysis (PCA) was performed based on the top 2000 highly variable genes. The first 20 principal components (PCs) were selected for downstream analysis using the Elbowplot function in the Seurat package. The FindClusters function in Seurat was utilized to identify main cell subgroups with the resolution set to the default value (res = 1). Subsequently, the t-SNE algorithm was employed to reduce the nonlinear dimensionality of the scRNA-seq sequencing data. Candidate markers for various cell subgroups were identified using the Seurat package, followed by cell annotation using the “SingleR” package. Cell communication analysis was conducted using the “CellChat” package in R language.
Lentivirus and cell transfection
The cDNA sequences were analyzed for potential short hairpin RNA (shRNA) target sequences based on GenBank. Two sequences targeting AURKA were designed, with one serving as a negative control without the knockdown sequence (sh-NC). The primer sequences were as follows: sh-NC (control) primer sequence: 5ʹ-GCGCGATAGCGCTAATAATTT-3ʹ, sh-AURK-1 primer sequence: 5ʹ-CCTGTCTTACTGTCATTCGAA-3ʹ, and sh-AURK-2 primer sequence: 5ʹ-CACATACCAAGAGACCTACAA-3ʹ. The oligonucleotides were synthesized by GenePharma® (Shanghai, China). A lentivirus packaging system was constructed using pLKO.1 (lentivirus gene silencing vector). The packaging virus and target vector were co-transfected into HEK293T cells (CRL-3216, ATCC, USA) using Lipofectamine 2000 (11668500, Thermo Fisher) at a cell confluence of 80–90%. The supernatant, which contained viral particles post-filtering and centrifugation, was collected 48 h after cultivation. Virus particles from cells in the exponential growth phase were harvested, and the viral titer was determined.
When the cells reached the logarithmic growth phase, they were detached using trypsin, resuspended to a concentration of 5 × 104 cells/mL, and then seeded into a 6-well plate at 2 mL per well. Prior to establishing the in vitro cell model, each lentivirus (MOI = 10, viral titer of 1 × 108 TU/mL) was added to the cell culture medium and incubated for 48 h. Stably transfected cell lines were selected using 2 µg/mL puromycin (UC0E03, Sigma-Aldrich, Germany) and maintained for two weeks.
The cell transfection groups were as follows: (1) sh-NC group: transfected with lentivirus carrying a negative control knockdown vector; (2) sh-AURKA-1 group: transfected with lentivirus carrying the sh-AURKA-1 vector; (3) sh-AURKA-2 group: transfected with lentivirus carrying the sh-AURKA-2 vector. All plasmids mentioned above were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.
In vitro cell culture and grouping
Human normal prostate epithelial cells PrEC (PCS-440-010, ATCC), CRPC cells PC-3 (CRL-1435, ATCC) and CRPC cells C4-2B (CRL-3315, ATCC) were cultured in RPMI 1640 supplemented with 1% penicillin–streptomycin and 10% FBS. The cells were maintained in a CO2 incubator (51032874, ThermoFisher, USA) at 5% CO2 and 37 ℃. The penicillin–streptomycin (15140148), FBS (10099), RPMI 1640 medium (11875101), and DMEM/F-12 medium (21041025) were all purchased from Gibco (USA). Aurora-A influence on cell sensitivity to DTX chemotherapy grouping: The sh-NC group was transfected with sh-NC cells and treated with the corresponding concentration of DTX (ZA040035, a reagent from China National Pharmaceutical Group Shanghai, China) for 48 h as a control. The sh-AURKA group was transfected with sh-AURKA cells and treated with the corresponding concentration of DTX for 48 h. The Vector group received an equal volume of DMSO water solution as the control group for Alisertib. The Alisertib group was treated with 100 nM Alisertib (MLN 8237, HY-10971, MedChemExpress) and 3 nM DTX for 48 h [45].
Cell Grouping for Nanoparticle Impact on Cells: PBS Group: Cells were treated with PBS for 48 h as a control. Alisertib Group: Cells were treated with 100 nM Alisertib for 48 h. DTX Group: Cells were treated with 3 nM Docetaxel (DTX) for 48 h. CM-AMS@D(+) Group: Cells were treated with 100 nM CM-AMS@D followed by photothermal irradiation for 5 min, then cultured for 48 h. CM-AMS@AD(+) Group: Cells were treated with 100 nM CM-AMS@AD followed by photothermal irradiation for 5 min, then cultured for 48 h.
γ-H2AX fluorescence imaging
Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by soaking the coverslips in 0.1% Triton X-100 for 5 min at room temperature. Subsequently, the cells were incubated overnight at 4 °C with the primary antibody Anti-γH2AX (ab81299, 1:250, Abcam, UK). After incubation, the cells were washed and further incubated with the secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor® 555) (ab150078, 1:1000, Abcam, UK) at room temperature for 1 h. The cell nuclei were stained with DAPI (4 µg/mL, 62248, Thermo Scientific) at room temperature for 30 min. Stained cells were observed under a confocal laser scanning microscope (Olympus Corporation) and analyzed using Image J.
Assessment of cell viability
Cell viability was assessed using Calcein AM (C3099, ThermoFisher, USA) and Propidium Iodide (P1304MP, ThermoFisher, USA) to distinguish live and dead cells. Cells were cultured with Calcein AM (1 μM) at 37 °C for 30 min, followed by washing with PBS three times. Subsequently, cells were incubated with PI (1 μM) for 10 min and washed again with PBS three times. Image acquisition was performed using a confocal microscope (880, Carl Zeiss AG, Germany), with each image captured from a different field of view. The experiment was conducted on three independent samples. Cell viability was calculated using ImageJ software (V1.8.0). Following the manufacturer’s instructions for the CCK-8 assay kit (C0041, Beyotime, Shanghai, China), cells were treated, and cell viability was measured by the CCK-8 method at 48 h post-culturing. For each measurement, 10 μL of CCK-8 detection solution was added, and after 4 h of incubation in a cell culture incubator, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.
Western blot
The total protein of the cell extract was lysed using RIPA lysis buffer (P0013B, Beyotime, Shanghai) containing 1% PMSF. The cell proteins were extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (78833, ThermoFisher Scientific, USA), and the total protein concentration of each sample was determined using the BCA assay kit (P0011, Beyotime, Shanghai). An 8–12% SDS gel was prepared according to the molecular weight of the target protein bands, and protein samples were loaded equally into each lane using a microvolume pipettor for electrophoresis separation. The proteins on the gel were transferred to a PVDF membrane (1620177, BIO-RAD, USA), followed by blocking with 5% skim milk at room temperature for 1 h. The membrane was then probed with anti-H2AX (1:1000, ab124781, Abcam), anti-gamma H2AX (ab81299, 1:2000, Abcam), anti-RAD51 (ab133534, 1:1000, Abcam), anti-Aurora A (ab108353, 1:1000, Abcam), anti-PARP (MA5-15031, 1:1000, ThermoFisher), anti-PSMA (ab133579, 1:1000, Abcam), and anti-beta Actin (ab8226, 1:2000, Abcam), and then incubated overnight at 4 °C. Subsequently, the membrane was washed three times with 1 × TBST at room temperature for 5 min each. The membrane was then incubated at room temperature with HRP-conjugated goat anti-rabbit IgG (ab6721, 1:2000) or goat anti-mouse IgG (ab6728, 1:2000) secondary antibodies for 1 h. After incubation, the membrane was washed three times with 1 × TBST buffer at room temperature for 5 min each. ECL detection reagent (1705062, Bio-Rad, USA) was then added, and the bands were exposed and imaged using the Image Quant LAS 4000C gel imaging system. Beta-Actin was used as an internal control, and the relative expression levels of the target proteins were analyzed using ImageJ software (V1.8.0.112) by calculating the ratio of the grayscale intensity of the target band to that of the reference band. The protein expression levels were evaluated by conducting three independent experiments for each group.
Isolation, activation, transduction, and culture of CAR-T cells
Primary human T cells were enriched by negative selection of unwanted cells from peripheral blood mononuclear cells (PBMC) (provided by Shandong Qilu Cell Therapy Engineering Technology) using the RosetteSep reagent kit (15025, Beijing Nuowei Biotech). Subsequently, the cells were cultured, and anti-CD3/anti-CD28 antibodies (11161D, Thermo Fisher, USA) were added to the T cell culture media (RPMI 1640, 10% FBS, 1% MEM NAA, 50 μM 2-mercaptoethanol, 300 IU/mL rhIL2). The primary T cells were activated for 24 h on day 0. CAR-T cells were constructed following the method by Alzubi et al., using PSMA-CAR lentivirus at a multiplicity of infection (MOI) of 10 and transduced into T cells with 4 μg/mL polybrene (sc-134220, Santa Cruz Biotechnology, USA). Flow cytometry was utilized employing fluorescein isothiocyanate (FITC) anti-human PSMA antibody (ab133579, Abcam), APC anti-human CD3 (ab239287, Abcam), PE anti-human CD4 (ab80590, Abcam), and FITC anti-human CD8 antibody (ab237709, Abcam) to identify the positive cell populations of T cells and CAR-T cells.
Preparation of AMS@AD NPs
*Abbreviation for the synthesized nanomaterials are listed in Table S1. Small Au NPs were synthesized by reducing HAuCl4·3H2O (G4022 Sigma Aldrich) using Tetrakis (hydroxymethyl) phosphonium chloride (THPC 404861 Sigma Aldrich). In a 45 mL aqueous solution 1 mol/L NaOH (0.5 mL 655104 Sigma Aldrich) was added followed by the addition of a mixture of 80% THPC solution (12 mL) and water (1 mL) to prepare a THPC solution (1 mL). The mixture was stirred for 5 min then the aqueous solution of HAuCl4·3H2O (1.5 mL 1 wt%) was rapidly added. Subsequently the solution was vigorously stirred for 15 min and stored at 4 °C for future use.
AMS NPs with a core–shell structure were synthesized using a co-biphasic (oil–water) layering system. Initially, 1.0 g of cetyltrimethylammonium bromide (CTAB, 1102974, Sigma Aldrich) and 100 mg of nano gold were redispersed in 60 mL of deionized water. Subsequently, CTAB was completely dissolved with continuous stirring at 60 °C. Then, 750 μL of 25% triethylamine was swiftly mixed into the aforementioned reaction system and vigorously stirred at 60 °C in an oil bath for 1 h. Simultaneously, a careful dropwise addition of a mixture of 20 mL of tetraethyl orthosilicate (12%, 131903, Sigma-Aldrich) and bis(triethoxysilyl) hexane (8%, 440574, Sigma-Aldrich) in cyclohexane (179191, Sigma-Aldrich) was added to the surface of the previously discussed aqueous solution. The mixture was gently stirred for 24 h, and after carefully separating the upper layer of cyclohexane using a pipette, core–shell AMS NP samples were obtained. After centrifugation and redispersion, the residue of CTAB was removed by alternate washing three times with water and ethanol (29221, Sigma Aldrich) at 60 °C in an oil bath for 12 h.
20 mg of AMS, 2 mg of DTX, and 4 mg of Alisertib were accurately weighed and mixed in 2 mL of deionized water. The mixture was stirred overnight at room temperature to prepare a solution of AMS@AD NPs. After centrifugation, the NPs were gently washed twice with deionized water to remove any free drugs.
Preparation of CM-AMS@AD
CAR-T cells were collected and subjected to multiple freeze–thaw cycles, followed by cell lysis under ice-cold conditions using sonication for 30 min. The supernatant was collected by centrifugation at 700g for 10 min at 4 °C, and the pellet was obtained by centrifugation at 14,000g for 30 min, yielding CAR-T cell membrane fragments. These fragments were mixed with AMS@AD, followed by sonication. The mixture was then extruded through a polycarbonate membrane (400 nm) 20 times to obtain AMS@AD NPs encapsulating CAR-T cells.
Analysis and characterization of NPs
Barrett-Joyner-Halenda (BJH) analysis of the N2 adsorption isotherm was conducted using a physical adsorption analyzer (Norcross, Micromeritics ASAP-2460, USA). The specific surface area, total pore volume, and average pore size distribution curve of the AMS nanoshell were determined by the BET method. The hydrodynamic size and zeta potential of the NPs were measured using a Zetasizer Nano ZS 90 (Malvern Instruments, UK). Transmission electron microscopy images were captured using a Hitachi H-7650 transmission electron microscopy instrument obtained from Shanghai Baihe Instrument Technology Co., Ltd. The sample was initially placed on a carbon-coated copper grid and negatively stained with phosphotungstic acid (PTA, 2% w/v, 496626, Sigma-Aldrich) for 10 s, followed by drying the reaction product. The morphology of the NPs was observed and captured at an accelerating voltage of 100 kV. The surface characteristics of the NPs were obtained using a scanning electron microscope (S-4800, Hitachi, Shanghai Fulei Optical Technology Co., Ltd). The concentration of AMS@AD was diluted to 0.05 wt% to prevent saturation during UV–visible spectrophotometry measurements using a UV–visible spectrophotometer (PS-200, Boreun Jingwei, China). The measurement wavelength range was set to 200–800 nm for analyzing the absorption spectra using UV Probe (Shimadzu) to identify characteristic absorption peaks. To test the stability of the prepared NPs, we incubated the NPs with PBS solution, 10% fetal bovine serum, and cell culture medium (21041025, Gibco, USA) for 48 h. At different time points, the changes in NP size were measured using dynamic light scattering. To observe the encapsulation of the cell membrane, AMS@AD was labeled with Dio (D275, Thermo Fisher) and the cell membrane was labeled with Dil (D282, Thermo Fisher). The fluorescence localization of CM-AMS@AD was observed using confocal laser scanning microscopy (CLSM).
Evaluation of the photothermal effect of nanomaterials
Nanomaterials were dispersed in solutions of varying concentrations, which were then aliquoted into a 96-well plate. The solutions were irradiated with a near-infrared laser at a wavelength of 887 nm at varying time intervals and intensities. The temperature of the solutions was measured using a thermocouple, and the temperature change of the dispersed solutions was recorded.
Cell uptake in monolayer culture models
C4-2B or PrEC cells at a density of 1 × 103 were seeded into a 96-well plate. The cells were then co-incubated with Dil-labeled AMS, AMS@AD, M-AMS@AD, and CM-AMS@AD for 4 h as experimental groups, while PBS was used as a control. Following co-incubation, the cells were fixed with a 4% (v/v) paraformaldehyde solution and stained with DAPI. Fluorescent signals of Dil and DAPI were observed using CLSM. Additionally, the fluorescence intensity of Dil in the cells of each group was quantitatively analyzed using flow cytometry.
Cellular uptake in spheroid culture models
This study investigates cellular uptake in C4-2B or PrEC cell spheroid models. C4-2B or PrEC cells were seeded onto round-bottom 96-well plates pre-coated with agarose gel and cultured under conditions similar to monolayer models. After 7 days of cultivation, the spheroids were treated with Dil-labeled AMS, AMS@AD, M-AMS@AD, and CM-AMS@AD and then incubated for 24 h. Subsequently, the spheroids were washed thrice with PBS and fixed with 4% (v/v) paraformaldehyde. Dil’s fluorescence signal was observed using the Z-stack function.
DC isolation
PBMCs were isolated using density gradient centrifugation (approved by the Animal Ethics Committee of Ganzhou Hospital-Nanfang Hospital. Monocytes were selected from PBMCs as precursors for DCs. These monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. The differentiation of DCs was induced by adding 100 ng/mL of G-CSF (PHC2035, Thermofisher), 250 U/mL of GM-CSF (AF-300-03-1MG, Thermofisher), and 80 ng/mL of IL-13 (200-13-1MG, R&D, Thermofisher) to the culture medium. The culture medium was half-replaced every 2 days. On the 7th day, the suspended cells were repackaged for further experiments, and the purity of DCs was assessed using anti-CD11c-FITC.
ATP content measurement
Cellular ATP content was determined by lysing cells using an ATP assay kit (BC0300, Solarbio, Beijing, China). According to the manufacturer’s instructions, ATP was extracted from the cells and measured using a UV spectrophotometer (DU720, Beckman, USA).
Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde (E672002, Sangon, China) for ~ 15–20 min, followed by treatment with 0.1% Triton X-100 (A110694, Sangon, China) for 10 min to permeabilize the cell membrane. Subsequently, cells were blocked with PBS containing 1% BSA for about 1 h to prevent nonspecific antibody binding. The cells were then incubated overnight at 4 °C with an anti-calreticulin (CRT) antibody (C7492, 1:200, Sigma-Aldrich, USA) or HMGB1 antibody (ab195010, 1:50, Abcam). Afterward, the cells were incubated with fluorescently labeled secondary antibodies (Sigma-Aldrich, USA) for 1 h, followed by observation and capture of CRT fluorescence signals using a fluorescence microscope. The observed images were analyzed using Image J.
Transwell co-culture
Transwell chambers (3422, 8 μm pore size, Corning, USA) were used for in vitro co-culture in a 24-well plate. Various nanomaterial treatments along with cancer cells (105/well) were seeded in the upper chambers of the Transwell system. DCs (105/well) were seeded in the lower chambers of the Transwell system and co-cultured with cancer cells for 24 h. The release of factors from the upper chambers and staining for CRT/HMGB1 were observed. Subsequently, cells from the lower chambers were collected, and DC differentiation and maturation states were investigated using flow cytometry.
Flow cytometry analysis
Flow cytometry was used to assess cell apoptosis rates. In brief, tumor cells (1 × 105/well) were collected and washed in chilled PBS, followed by staining with a detection kit (APOAF-20TST, Sigma-Aldrich, USA) using propidium iodide in the dark for 15 min. Subsequently, the pellet was resuspended in 400 μL of binding buffer and stained with 5 μL of Annexin-V as provided in the kit. Cells were then analyzed using a flow cytometer. Cells in the upper right quadrant represented by Annexin V + PI+ phenotype indicated late apoptotic cells, while cells in the lower right quadrant with Annexin V + PI− phenotype corresponded to early apoptotic cells. Cells in the upper left quadrant with Annexin V-PI + phenotype indicated necrotic cells, and cells in the lower left quadrant with Annexin V-PI− phenotype represented viable cells.
Multi-color flow cytometry was utilized to analyze the immune cell composition in tumors, lymph nodes, and spleens. Mouse samples from each group were collected and digested in HBSS buffer (24020117, ThermoFisher Scientific) containing 0.5 mg/mL type IV collagenase and 0.25 mg/L DNase I at 37 °C for 30 min. After digestion, the samples were filtered through a 40 μm cell strainer and centrifuged at 400g for 10 min. Cells were first incubated with Fc-blocking antibodies (BioLegend) for 15 min to prevent nonspecific binding, followed by staining with monoclonal antibodies conjugated with fluorescent dyes for cell surface markers. The antibodies used were as follows: FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE (320027), CD45 PerCP/Cy5.5 anti-human (304028), CD8 Brilliant Violet 650 anti-human (344730), CD3 PE/Cy7 anti-human (300316), CD11C PE (980602), CD86 FITC (374203), CD80 APC (375403), CD44 FITC (397517), CD62L APC (980706). These antibodies were purchased from BioLegend. After staining, the samples were washed with PBS containing 1% BSA and then resuspended in 500 μL of PBS. Flow cytometry analysis was conducted using the BD FACSAria Fusion Flow Cytometry Cell Sorter (BD Biosciences), and data analysis was performed using FlowJo v.10 software (FlowJo LLC).
Flow cytometry was used to analyze the cell cycle. Initially, the transfected cells were digested with trypsin and centrifuged at 300g for 5 min. After washing twice with PBS, the cells were fixed overnight in 75% ethanol at − 20 °C. Prior to analysis, the cells were washed twice with PBS and then incubated in the dark at room temperature for 15 min with 10 μL of 5 mg/mL PI stock solution in 500 μL of 1 × PBS to achieve a final concentration of 50 μg/mL. The BD FACSCanto II flow cytometer (BD Biosciences, USA) was used for cell cycle analysis, and FlowJo software was employed for data analysis.
Animal experiments
All animal studies were conducted in compliance with the guidelines outlined in our institution’s “Guidelines for the Care and Use of Laboratory Animals.” Four-week-old female humanized huHSC-(M-NSG) mice (Catalog No: NM-NSG-017, Shanghai Southern Model Biological Technology Co., Ltd) were utilized to establish a castration-resistant cancer model.
C4-2B (or C4-2B-LUC) cells (2 × 106) were injected subcutaneously (sc) into the left flank of mice to establish a prostate xenograft model. Fluorescence signals in the mice were monitored using the CRi Maestro in vivo imaging system (CRi Inc., USA). Seven days after injection, tumor size was measured, and eligible mice were randomly assigned to different groups. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of our institution. Nanoparticle solutions (0.10 mL) were administered via tail vein injection every 4 days, and photothermal therapy (PTT) was performed 24 h after injection. On day 28, the mice were euthanized.
The mouse groups were as follows: Ctrl Group: Tumor-bearing mice received 0.10 mL of PBS via tail vein injection without PTT, serving as a positive control. Alisertib Group: Tumor-bearing mice received 0.10 mL of Alisertib solution via tail vein injection without PTT. DTX Group: Tumor-bearing mice received 0.10 mL of Docetaxel (DTX) solution via tail vein injection without PTT. CM-AMS@D(+) Group: Tumor-bearing mice received 0.10 mL of CM-AMS@D solution via tail vein injection, followed by PTT. CM-AMS@AD(+) Group: Tumor-bearing mice received 0.10 mL of CM-AMS@AD solution via tail vein injection, followed by PTT.
Tumor growth was monitored every 4 days using non-invasive bioluminescence imaging. On day 28, mice were euthanized by cervical dislocation under deep anesthesia with isoflurane (R510-22-10, RWD, Shenzhen, China).
For the imaging of excised tissues, tumors and major organs, including the heart, liver, spleen, lungs, and kidneys, were removed. These organs or tumors were then isolated and processed for subsequent experimental analysis using Formalin-Fixed Paraffin-Embedded (FFPE) or rapid freezing. Additionally, blood samples were collected for biochemical analysis, while blood, spleen, and tumor samples were collected for flow cytometry analysis.
Detection of biochemical parameters
Biochemical parameters in the serum of mice were measured using alanine transaminase (ALT) activity assay kit (E1010, Sigma-Aldrich, USA), aspartate transaminase (AST) activity assay kit (E1020, Sigma-Aldrich, USA), blood urea nitrogen (BUN) assay kit (60-1100, BioAssay Systems, USA), and creatinine (CRE) assay kit (CR200, RanDTX Laboratories Ltd, UK). The specific procedures were conducted following the instructions provided with each assay kit.
Enzyme-linked immunosorbent assay (ELISA)
The levels of INFγ and TNF-α in the serum of mice were determined using Mouse INFγ ELISA Kit (MIF00, R&D Systems, USA) and Mouse TNF-α ELISA Kit (MTA00B, R&D Systems, USA). The assays were performed according to the instructions provided with each kit.
Pathological histological staining
Cell apoptosis in tumor tissue paraffin sections was detected using the TUNEL staining kit. Tumor tissue 6 μm paraffin sections were deparaffinized and rehydrated. Subsequently, the tissue sections were incubated at room temperature for 20 min in Tris buffer solution (pH = 8) containing 15.3 mg/mL proteinase K, followed by rinsing with 50 mM TBS (pH 7.6). The sections were then incubated with a green fluorescent enzyme solution (C1086, Beyotime, Shanghai, China). After TUNEL labeling, cell nuclei were stained with DAPI. Apoptotic cells appeared as green fluorescence, observed using CLSM (IX73, Olympus Corporation, Japan), and image processing and quantitative analysis of TUNEL-positive cells were performed using ImageJ.
Tumor tissues were fixed with formalin and prepared as paraffin sections, following the standard procedures of immunohistochemical staining. Rabbit anti-γ‐H2AX (ab81299, 1:1000, Abcam, UK), rabbit anti-Ki67 (ab16667, 1:200, Abcam), and rabbit anti-RAD51 (ab133534, 1:1000, Abcam, UK) antibodies were applied overnight in a dark chamber. The next day, goat anti-rabbit IgG (ab6721, 1:1000, Abcam) secondary antibodies were incubated for 30 min, followed by incubation with Streptavidin–Biotin Complex (SABC, Vector Corporation, USA) at 37 °C for 30 min. DAB chromogenic kit (P0203, Beyotime Biotechnology, Shanghai) was used by adding a drop of chromogen to the specimen, staining for 6 min, and counterstaining with hematoxylin for 30 s. The sections were dehydrated in a series of ethanol concentrations (70%, 80%, 90%, 95%, and absolute ethanol) for 2 min each, followed by two immersions in xylene for 5 min each for transparency. Finally, the sections were mounted with neutral resin and observed under a brightfield microscope (BX63, Olympus, Japan). ImageJ software was used to quantify the average grayscale value (staining intensity) and the number of positive cells. Statistical analysis was conducted to compare differences between different groups, visualizing the results to quantify the staining effect on cells or tissue sections.
Hematoxylin and eosin (H&E) staining: Tissue samples were obtained for examination and subjected to fixation. After sectioning the paraffin blocks, the wax was removed in xylene, followed by dehydration in 100%, 95%, and 70% ethanol, and eventually rinsing with water. The prepared sections were immersed in hematoxylin staining solution (H8070, Solarbio, Beijing, China) and stained for 5–10 min at room temperature. Subsequently, the sections were rinsed with distilled water, dehydrated in 95% ethanol, and placed in an eosin staining solution (G1100, Solarbio, Beijing, China) for 5–10 min, followed by standard dehydration, clearing, and mounting.
Statistical analysis
Our study utilized R language version 4.2.1, with RStudio as the integrated development environment for compiling R code, using RStudio version 2022.12.0-353. All data were processed using GraphPad Prism 8.0. Descriptive data were presented as mean ± standard deviation (mean ± SD). Non-paired t-tests were employed for the comparison between the two groups, while one-way analysis of variance (ANOVA) was utilized for comparisons among multiple groups. Levene’s test was performed to assess the homogeneity of variance. In cases of homogeneity of variance, Dunnett’s T3 and LSD-t tests were conducted for pairwise comparisons. When variance was not homogeneous, Dunnett’s T3 test was used. A significance level of P < 0.05 was considered statistically significant for differences between groups.
