Sensitizers
Sinoporphyrin sodium (DVDMS, molecular formula: C68H66N8O9Na4, molecular weight: 1230.265 Da, purity: 98.5%) was produced by Jiangxi Qinglong High Technology Co., Ltd. (Yichun, China), dissolved in 0.9% sodium chloride to form a solution with a final concentration of 1.25 mg/mL, sterilized by a 0.22-μm filter, and stored in the dark at −20 °C. The chemical structure and absorption spectrum of DVDMS are presented in Fig. 1A, B.
Cell culture
The human GC cell lines HGC27 and AGS were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and the human GC cell line MGC803 and human gastric epithelium cell line GES-1 were obtained from the Key Laboratory for Tumor Precision Medicine of Shaanxi Province (Xi’an, China). HGC27 and MGC803 cells were cultured in the RPMI 1640 medium (Sigma-Aldrich, WI, USA), AGS cells were maintained in Ham’s/F-12 medium (Procell, Wuhan, China), and GES-1 cells were grown in Eagle’s minimum essential medium (DMEM, Sigma-Aldrich, WI, USA), containing 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillin/streptomycin (New Cell & Molecular Biotech, Suzhou, China). All cells were incubated in a water-saturated atmosphere of 5% CO2 at 37 °C and were collected at the peak of the logarithmic growth phase for experiments.
Reagents and irradiation
The Cell Counting Kit-8 (CCK-8) assay kit was purchased from TargetMol (MA, USA), 5-ethynyl-2′-deoxyuridine (EdU) assay kit was from Beyotime (Shanghai, China), N-acetylcysteine (NAC) was from Selleck (Shanghai, China), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was from KeyGen BioTECH (Nanjing, China). The Annexin V-FITC/PI Apoptosis detection kit was purchased from BD Bioscience (Franklin Lakes, NJ, USA), chloroquine (CQ) phosphate salt was from Sigma-Aldrich (Germany), and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was from Solarbio (Beijing, China). We obtained 3-methyladenine (3-MA), LY294002, and 740 Y-P from MedChemExpress (Monmouth Junction, NJ, USA). All the other reagents were analytical grade commercial products.
Semiconductor lasers (excitation wavelength: 450 nm and 630 nm, respectively; manufacturer: Blueray Medical Ltd., Xi’an, China) were carried out as the source for the evocation of the photodynamic effect.
Detection of intracellular DVDMS uptake
All cell lines (HGC27, MGC803, AGS, and GES-1) were planted in 24-well plates (1 × 105 cells/well) and 6-well plates (3 × 105 cells/well), respectively, and incubated with DVDMS (1 μM) in the dark at 37 °C for indicated time periods. For measurement of the intracellular uptake of DVDMS, cells were collected at different incubation time points (0, 1, 2, 4, 6, 8 h) and detected with flow cytometry (FACSCalibur, BD, USA) and fluorescence microscopy (Olympus, Japan). The mean fluorescence intensity (MFI) of DVDMS in cells was recorded under uniform detection conditions.
Cell viability assay
Cell viability was estimated using CCK-8, colony formation, and EdU assays. The GC cell lines HGC27, MGC803, AGS, and gastric epithelium cell line GES-1 were cultured in 96-well plates (5 × 103 cells/well) and incubated with DVDMS at various concentrations (0, 0.05, 0.1, 0.2, and 0.4 μM) at 37 °C in darkness for 4 h and then exposed to an energy density of 10 mW/cm2 in a BL (wavelength 450 nm) for 600 s to achieve a final laser dose of 6 J/cm2. Meanwhile, DVDMS alone with the same concentration gradient was incubated separately to detect the cytotoxicity. After PDT for 24 h, CCK-8 was mixed with each well to detect cell viability and GraphPad Prism 8.0 software was performed to calculate the IC50. Notably, the IC50 of each cell line was selected as the appropriate incubation concentration of DVDMS in subsequent experiments. And then, the PDT effects of BL (450 nm) and RL (630 nm), or DVDMS and 5-aminolevulinic acid (5-ALA) on cells were compared under the same drug dose (IC50) and irradiation power (6 J/cm2), and morphological changes in the cells were observed under a microscope. In addition, the light dose correction (LDC) of BL and RL should be considered according to the method proposed by Schaberle [28].
A colony formation assay was carried out to assess the long-term proliferative potential of all GC cell lines after PDT. Meanwhile, the maximum IC50 of GC cell lines was used as the incubation concentration of DVDMS to observe the PDT effect on the proliferation of normal gastric mucosal cells. The treated cells were seeded in 6-well plates (600 cells/well) and cultured for 10 days. During this period, the culture medium was replaced with a fresh medium every 3 days. After visible colonies formed, cells were fixed in 4% paraformaldehyde (PFA) for 10 min and incubated with 0.1% crystal violet solution for 20 min. The number of stained colonies containing more than 50 cells was observed and manually counted under a microscope.
For the EdU assay, cells were seeded in 24-well plates at a density of 1 × 105 cells/well. After PDT, the cells were incubated with EdU working solution for 2 h, fixed with 4% PFA, and permeated with 0.3% Triton X-100 PBS. And then, a series of reaction solutions were added to the well and the nuclei were finally stained with Hoechst. After that, the results of cell proliferation were visualized by a fluorescence microscope.
Measurement of intracellular ROS production
Briefly, cells were incubated with serum-free medium containing 10 μM DCFH-DA in the dark for 30 min prior to PDT. After treatment, the fluorescence intensity was observed immediately with a fluorescence microscope and then detected by flow cytometry. In addition, an electron spin resonance (ESR) spectrometer (A300-9.5/12, Bruker, Germany) was used to detect the different types of ROS, and 2,2,6,6-tetramethyl-4-piperidone (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were selected as capture agents to identify singlet oxygen (1O2) and hydroxyl radicals (·OH), respectively.
For inhibitory experiments, cells were preincubated with 5 mM NAC (a ROS scavenger) for 1 h and washed with PBS three times after 1 h of PDT. Then, the fluorescence intensity and cell viability were assessed using the same method as above.
RNA sequencing
According to the instructions of Novogene Co., Ltd. Tianjin Sequencing Center, the total RNA extracts of HGC27 and MGC803 cells treated with BL-PDT were separated with TRIzol, isolated by chloroform, and extracted using ethanol, respectively. The cDNA library construction, library purification, and transcriptome sequencing were then carried out for deep data analysis. Of note, three duplicate samples from each group were used for RNA-sequencing analysis.
Apoptosis assay
The apoptotic ratios of HGC27, MGC803, and AGS cells treated with BL-PDT were detected via flow cytometry. The cells were respectively implanted into 6-well plates at a density of 3 × 105 cells/well. After BL-PDT for 24 h, the cells were washed twice with precooled PBS, collected in 1.5-mL tubes, and centrifuged at 500 g (4 °C) for 5 min. Subsequently, the cells were resuspended in 100 μL of a binding buffer and mixed with 4 μL of Annexin V-FITC and 4 μL of propidium iodide (PI) for 15 min. After that, 400 μL of binding buffer was added for dilution and flow cytometry was performed to measure the proportions of apoptotic cells.
Transfection and evaluation of fluorescent dots
According to the instructions of Hanbio Co., Ltd. (Shanghai, China), all GC cells were seeded in 6-well plates at a density of 20 × 105 cells/well overnight. The appropriate concentrations of mRFP-GFP-LC3 adenovirus and polyethyleneimine were mixed and incubated for 20 min at room temperature and then slowly added to the serum-free medium, which was used to replace the original cell culture medium. After 24 h of transfection, the fresh serum medium was replaced. After 4 h of stabilization, the cells were digested by trypsin and planted in 24-well plates containing climbing sheets. After the growth of cell adhesion, PDT was performed. Then, the cells were incubated in the dark at 37 °C for 24 h, observed under a confocal fluorescence microscope, and analyzed by ImageJ software. In merged images, yellow fluorescent spots in transfected cells demonstrated autophagosomes, red spots indicated autophagic lysosomes, and the enhancement of both yellow and red fluorescent dots indicated increased autophagic flux.
Transmission electron microscopy (TEM) analysis
After treatment with BL-PDT for 12 h, the cells were harvested and fixed with ice-cold 2.5% glutaraldehyde for 2 h at 4 °C. Then, the fixed cells were post-treated with 1% buffered osmium tetroxide for 1 h at 4 °C and dehydrated by subsequently passing through a graded series of ethanol. After fixation and embedding, the cell samples were sectioned into thin pieces, stained with 3% uranyl acetate/lead citrate, and finally visualized with an H-7650 TEM (Hitachi, Japan).
Western blot assay
The expression levels of involved proteins were measured by western blotting analysis and analyzed by ImageJ software. Briefly, the cell samples at 24 h after BL-PDT were collected and lysed in 1 × radioimmunoprecipitation assay buffer with 1% phenylmethylsulfonyl fluoride for 20 min on ice. After quantification by a bicinchoninic acid assay kit (Beyotime, Shanghai, China), lysates were boiled for 10 min at 100 °C, and aliquots of 20 μg of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes that were then blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies on a shaker (4 °C, overnight). After 3 times of washing with TBST and 2 h of incubation with the corresponding secondary antibody at room temperature, the immunoblots on the membranes were observed using an enhanced chemiluminescence detection kit (Millipore, Burlington, MA, USA). The primary antibodies included LC3 and PARP antibodies (all from Cell Signaling Technology, USA); p62, Beclin-1, Bax, Bcl-2, Caspase 3, and Caspase 9 antibodies (all from Abcam, UK); and PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR antibodies (all from Abmart, China). Mouse anti-human β-Actin antibody and goat anti-mouse or anti-rabbit secondary antibody were purchased from Abcam.
Animal experiment
BALB/c mice (age 4–5 weeks, male, 18–20 g body weight) were purchased from the Shanghai Experimental Animal Center of the Chinese Academic of Sciences (Shanghai, China) and housed in an air-conditioned animal center at 22 °C ± 2 °C with free access to food and water. All in vivo experimental procedures were approved by the ethics committee of Xi’an Jiao Tong University (Xi’an, China). After 1 week of adaptation, 6 × 106 MGC803 cells were subcutaneously injected into the right dorsal region of BALB/c mice. The body weight and tumor size of each animal were measured every other day, and the total volume was calculated as V = (length × width2)/2. When tumors reached an average volume of 100 mm3 (approximately 10–12 days post-injection), the mice were randomly divided into four groups: (1) control (0.9% saline, 0.1 mL), (2) 450-nm laser (60 J/cm2), (3) DVDMS (2 mg/kg, 0.1 mL), and (4) PDT (DVDMS + 450-nm laser). DVDMS was injected into the caudal vein, and irradiation was performed 24 h after injection [18]. Fourteen days after treatment, all the mice were sacrificed, and the entire tumors were collected and weighed.
In addition, the effects of 450-nm and 630-nm laser-mediated DVDMS-PDT on GC growth were also compared in vivo. The mice were randomly divided into three groups: (1) DVDMS (2 mg/kg, 0.1 mL), (2) RL-PDT (DVDMS + 630-nm laser (60 J/cm2)), and (3) PDT (DVDMS + 450-nm laser (60 J/cm2)), and the remaining steps were the same as above.
Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis
After being fixed in 4% PFA for at least 24 h, the tumor tissue and major organs of each animal were embedded in paraffin, sliced into thick sections, and stained with H&E. Histopathological changes were observed with an inverted fluorescence microscope. For IHC examination, sliced tumor tissues were incubated with primary antibodies (LC3-II, p-Akt, and Ki-67) for 20 h at 4 °C and secondary antibody for 1 h. After visualization with diaminobenzidine solution conjugated with hematoxylin, the images were observed with a microscope and analyzed by ImageJ software.
Statistical analysis
All quantitative results are expressed as the mean ± standard deviation (SD). Statistical analysis was carried out with GraphPad Prism 8.0 software by the two-tailed unpaired Student’s t test and log-rank test. Statistical significance is expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.