The human CRC cell lines HCT116 and SW480, as well as the mouse CRC cell lines CT26 and MC38, were obtained from the Chinese Academy of Sciences’ Cell Bank of Type Culture Collection (Shanghai, China). HCT116 cells were cultured in the RPMI 1640 medium (Sigma-Aldrich, St.Louis, MO,USA), while SW480, CT26 and MC38 cells were maintained in Eagle’s minimum essential medium (DMEM, Sigma-Aldrich, St.Louis, MO,USA), supplemented with 10% fetal bovine serum (Biological Industries, Jerusalem, Israel). In addition, the culture medium was supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (New Cell & Molecular Biotech, Suzhou, China). All cell lines were validated by STR profiling and tested negative for mycoplasma. Cells were incubated in a humidified atmosphere of 5% CO${}_2$ and 21% O${}_2$ at 37 °C and were gathered for the tests during the logarithmic growth phase.

The sensitizer ATPP-DTPA, amino derivative of tetraphenyl porphyrin with diethylenetriaminepentaacetic acid (DTPA), was provided by Institute of Biomedical Engineering, CAMS&PUMC, molecular weight: 1004 Da, purity: 98.5%. ATPP-DTPA was dissolved in phosphate-buffered saline (PBS) to form a stock solution with a final concentration of 1 mM. 5-ALA was purchased from Suzhou NMT Biotech Co., Ltd., dissolved in Dimethyl sulfoxide (DMSO) to form a stock solution with a final concentration of 50 mM. The sensitizers were sterilized with 0.22-µm filter membrane, then stored in darkness at –20 °C and used within one month.

Working solution of ATPP-DTPA was fresh prepared at various concentrations of 0, 0.625, 1.25, 2.5, 5, 10 µM and 5-ALA was also fresh prepared with concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8 mM. The PDT light source was provided by the semiconductor lasers (excitation wavelength: 450 nm; manufacturer: Blueray Medical Ltd., Xi’an, China). Cells were first incubated with ATPP-DTPA or 5-ALA under darkness for 4 hours and then treated with laser irradiation of 20 mW/cm${}^2$ for 10 min. After PDT, cells were returned to incubated at 37 °C with 5 % CO${}_2$ until further analysis.

For inhibitory experiments,10 µM Z-VAD-FMK and SB202190 from MedChemExpress (Monmouth Junction, NJ, USA) were incubated with CRC cells 1 h before irradiation to inhibit apoptosis signal pathway and p38 MAPK signal pathway, respectively.

HCT116 and SW480 cultured in 6-well plates (3 $\times$ 10${}^5$ cells/well) were incubated with ATPP-DTPA (10 µM) under darkness at 37 °C for indicated time periods (0, 1, 2, 4, 8, and 12 hours). Following PBS washing, cells were harvested at various incubation times and identified by flow cytometry (BD FACSCalibur, Franklin Lakes, NJ, USA). The data were then processed and presented in a line graph.

The Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2${}^{\prime }$-deoxyuridine (EdU) were used to measure the vitality of the cells. The CCK-8 kit was acquired from TargetMol (Waltham, MA, USA), while the EdU assay kit was bought from Beyotime (Shanghai, China). For the CCK-8 assay, four cell lines—HCT116, SW480, CT26, and MC38—were initially cultivated in 96-well plates (5 $\times$ 10${}^3$ cells/well) and then treated with ATPP-DTPA and 5-ALA at different concentrations mentioned above. After four hours incubation, PDT was performed. In order to determine its dark cytotoxicity, 5-ALA and ATPP-DTPA were incubated independently with the same concentration gradient without illumination. Following avoiding from light for 24 hours, CCK-8 was introduced to each well to assess cell survival. At the meantime, in order to establish the appropriate incubation dose for the subsequent ATPP-DTPA studies, the IC${}_{50}$ was calculated by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). For EdU assay (Beyotime, Shanghai, China), the cells were inoculated at a density of 3 $\times$ 10${}^5$ cells per well in a 24-well plate. Following PDT, 0.3% TritonX-100 PBS was penetrated and paraformaldehyde (PFA) was fixed after two hours of incubation with EdU working solution. Additional reaction solutions were added to the well to complete the final reaction process. Ultimately, cell multiplication was discovered using a fluorescent microscope.

The level of total intracellular ROS was measured by 2${}^{\prime }$,7${}^{\prime }$-dichlorodihydro-fluorescein diacetate (DCFH-DA). DCFH-DA was purchased from KeyGen BioTECH (Nanjing, China). For thirty minutes prior to PDT, HCT116 and SW480 cells were cultured in a serum-free media containing 10 µM DCFH-DA. Following the treatment, cells were washed twice with PBS, then, the fluorescence microscope and flow cytometry were used to measure the intensity of ROS fluorescence intensity.

For inhibitory experiments, N-acetylcysteine (NAC) from Selleck (Shanghai, China) was added to the medium with a concentration of 10 mM and incubated with the cells for 2 hours before PDT to played its role of ROS scavenger.

The apoptosis rate of HCT116 and SW480 was detected by flow cytometry. The supplier of the Annexin V-FITC/PI Apoptosis kit was BD Bio-science (Franklin Lakes, NJ, USA). At a density of 5 $\times$ 10${}^5$ cells per well, HCT116 and SW480 cells were initially inserted into 6-well plates to perform PDT. After 24 hours of PDT, cells were washed twice using PBS and then collected into 1.5 mL-tubes. After centrifugation, cells were then suspended in 100 µL 1 $\times$ B-D buffer, followed by 3 µL Annexin V-FITC and 3 µL propidium iodide (PI) staining for 15min avoiding light. Finally, flow cytometry was used to evaluate and calculate the apoptotic rate.

HCT116 cells were divided into ATPP-DTPA group and PDT group with three independent repetitive sample experiments. After 24 hours of PDT to HCT116 cell, TRIzol was used to separate the total RNA extracts. The samples were sent to Novogene Co., Ltd. Tianjin Sequencing Center for test.

Cells were inoculated in 6-cm cell culture dish, when the cell density reached about 70%, PDT and other control treatments were performed. We first collected the cell samples at 12 hours after PDT. Then the samples were lysed using 1 $\times$ Protease Inhibitor Cocktail in RIPA lysis buffer (New Cell & Molecular Biotech, Suzhou, China) for 15 min on ice. After centrifuging cell lysates at 13,000 $\times$g for 15 minutes at 4 °C, the supernatant was collected and heated to 100 °C for 10 minutes. Quantification of protein samples by bicinchoninic acid assay (BCA) determination and aliquots of 30 µg of protein were loaded onto 10% or 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene difuoride (PVDF) membranes. The membranes were blocked with 5 % non-fat milk for 1 h at room temperature. Later on, they were incubated overnight at 4 °C on a shaker with the following primary antibodies against: poly ADP-ribose polymerase (PARP) obtained from CST (Cell Signaling Technologies, Danvers, MA, USA, 1:1000, #9532), Cleaved Caspase-9 (1:1000, #9509, CST), p38 MAPK (1:1000, #8690, CST), Phospho-p38 MAPK (1:1000, #4511, CST), Vinculin obtained from Abmart (Shanghai, China, 1:1000, T55164), Caspase-3 (1:1000, T55051, Abmart), Caspase-9 (1:1000, T55049, Abmart). Subsequently, the membranes were incubated with the corresponding secondary antibody (1:5000, M21001, M21002, Abmart) at room temperature for 1 h. Finally, the immunoblots on the membranes were observed using an enhanced chemiluminescence detection kit (Millipore, Burlington, MA, USA).

Aged 4–5 weeks male Balb/C nu/nu mice (Shanghai Experimental Animal Center of the Chinese Academic of Sciences, Shanghai, China) were fed under standard conditions and prepared for in vivo experiment. The Xi’an Jiaotong University Ethics Committee (Xi’an, China) gave its approval to all in vivo tests, which were conducted in accordance with the rules set out by the National Laboratory Animal Care and Use Institute. Mice were received a subcutaneous injection of 5 $\times$ 10${}^6$ HCT116 cells into their right dorsal area. After the injection, the tumor took ten days to develop to a volume of approximately 100 mm${}^3$, then a total of 24 mice were randomly divided into four groups: (1) control group (0.1 mL PBS for control), (2) blue laser group (tumors were exposed to 450-nm blue laser with a light dose of 100 mW/cm${}^2$ for 10 min, about 60 J/cm${}^2$), (3) ATPP-DTPA group (5 mg/kg, 0.1 mL ATPP-DTPA was injected), (4) PDT group (ATPP-DTPA + 450-nm blue laser). ATPP-DTPA was injected through the tail vein and laser therapy was performed after one day of avoiding light. Every two days, the tumor size of each animal was measured using vernier calipers, and the total volume was computed using the formula V = (length $\times$ width${}^2$)/2. After two weeks, all the mice were sacrificed and the tumors were collected for follow-up experiments.

All animal’s main organs and tumor tissues were preserved in 4% paraformaldehyde (PFA) for no less than 24 hours. Samples were cut into 5-µm-thick slices, and hematoxylin and eosin (H&E) followed by addition. The histopathological changes were observed by inverted fluorescence microscope. Primary antibodies (cleaved-PARP, phospho-p38 MAPK, and Ki-67) were incubated on the sections for 16 hours at 4 °C and goat anti-rabbit secondary antibody was followed for 1 h at room temperature, then visualized using diaminobenzidine (DAB) solution (34002, Thermo Fisher Scientific, Waltham, MA, USA). Finally, we observed the image with a microscope and analyzed it with Image J 1.46r software (National Institute of Health , Bethesda, MD, USA).

Each data was repeated in three independent experiments, and the mean $\pm$ standard deviation (SD) was employed to express all quantitative results. The t-test and logarithmic rank test were employed in the statistical analysis, which was conducted using GraphPad Prism 8.0 software. The statistical significance is expressed as *p 0.05, **p 0.01, and ***p 0.001.

ATPP-DTPA, as a new photosensitizer, has the basic structure of porphyrin rings (Fig. 1A). The absorption spectrum of ATPP-DTPA in PBS was showed in Fig. 1B, whose highest absorption peak was around 410-nm. Since ATPP-DTPA has a pretty absorption in the blue light spectrum, 450-nm blue laser can be selected as the light source for subsequent PDT. Next, we measured the mean fluorescence intensity (MFI) by flow cytometry to evaluate the intracellular uptake of ATPP-DTPA. We could observe that the accumulation of ATPP-DTPA in CRC cells (HCT116 and SW480) exceeded 90% after 4 hours of incubation, and with the extension of incubation time, the accumulation did not continue to increase significantly (Fig. 1C,D). Therefore, four-hour incubation of ATPP-DTPA was selected for our further experiments.

Fig. 1.

The properties of 5-(4-aminophenyl)-10,15,20-triphenylporphyrin with diethylene-triaminopentaacetic acid (ATPP-DTPA). (A) Basic chemical structural formula of ATPP-DTPA. (B) The absorption spectrum of ATPP-DTPA in PBS. (C,D) The intracellular uptake of ATPP-DTPA in HCT116 and SW480 cells were analyzed by flow cytometry at different periods (n = 3, mean $\pm$ standard deviation (SD)).

The CCK-8 assay was utilized to explore whether ATPP-PDT could affect the viability of CRC cells. As indicated in Fig. 2A, PDT substantially decreased the proliferation of CRC cells in both human (HCT116 and SW480) and mouse (MC38 and CT26) CRC cell lines. It’s worth noting that the reduction in CRC cell proliferation was observed in an ATPP-DTPA dose-dependent manner. The corresponding concentration of ATPP alone without irradiation did not show obvious cytotoxic effect on the cells. According to the data of cell survival rate, we calculated the IC${}_{50}$ of each cell line (HCT116: 5.966 µM; SW480: 5.500 µM; MC38: 5.461 µM; CT26: 7.012 µM), as the concentration basis for follow-up experiments. The morphological changes after ATPP-PDT were also recorded by light microscope (Supplementary Fig. 1A), and chromatin contraction and nuclear rupture could be observed in PDT group. Of note, under the same circumstances, we also conducted a comparison with the traditional photosensitizer 5-ALA (Supplementary Fig. 1B), and ATPP showed a better PDT effect compared with the ALA (IC${}_{50}$ of HCT116: 1.140 mM; SW480: 0.496 mM; MC38: 1.081 mM; CT26: 1.144 mM). EdU assay further verified the anti-proliferation effects of ATPP-PDT on CRC cells (Fig. 2D and Supplementary Fig. 2). As paired with flow cytometry, Annexin V-FITC/PI double labeling revealed a significant increase in apoptotic cells in PDT group as compared to other groups, suggested that there might be a relationship between PDT-mediated growth inhibition and apoptosis of CRC cells (Fig. 2B,C). These data indicated that ATPP-PDT had a strong phototoxic effect on CRC cells.

Fig. 2.

Anti-proliferative effects of ATPP-photodynamic therapy (PDT) against colorectal cancer (CRC) cells. (A) Photodynamic effects and cytotoxic of ATPP-DTPA on CRC cells (n = 3, mean $\pm$ SD). Flow cytometry analysis of the apoptosis (B), corresponding mean fluorescence intensity (MFI) (C), and 5-ethynyl-2${}^{\prime }$-deoxyuridine (EdU) assay (D) in HCT116 and SW480 cells after ATPP-PDT for 24 h (n = 3, mean $\pm$ SD). Scale bar = 50 µm. **p 0.01, and ***p 0.001. PI, Propidium iodide.

Numerous studies have demonstrated direct correlations between the high levels of intracellular ROS generation and the cell damage caused by PDT. Therefore, we detected the ROS generation during the process of ATPP-PDT, fluorescence microscopy suggesting that considerably increased ROS was observed in PDT group, whereas the amount of ROS decreased significantly after adding NAC, a classical ROS scavenger (Fig. 3A,C). Meanwhile, results of flow cytometry (Fig. 3B) further confirmed the same conclusions. Functionally, as shown in Fig. 3D and Supplementary Fig. 3, NAC could significantly mitigate the inhibitory impact of PDT on cell survival and proliferation rate, indicating that ROS plays a critical role in regulating the inhibition of cell growth during PDT.

Fig. 3.

Production of reactive oxygen species (ROS) in ATPP-PDT and its effect on CRC cells. (A) Representative fluorescence images of intracellular ROS generation in HCT116 and SW480 cells. Scale bar = 50 µm (n = 3, mean $\pm$ SD). Flow cytometry analysis (B) and corresponding MFI (C) of the content of ROS in each treatment group with or without N-acetylcysteine (NAC) (incubation for 2 h) (n = 3, mean $\pm$ SD). (D) EdU assay of HCT116 and SW480 cells after adding NAC (incubation for 2 h). Scale bar = 50 µm. ***p 0.001.

We have observed in Fig. 2B that ATPP-PDT-mediated growth inhibition of CRC cells is related to apoptosis by flow cytometry. To further verify our conclusion, we carried out western blotting experiment (Fig. 4A and Supplementary Fig. 4A). Compared with other groups, ATPP-PDT elevated the expression of cleaved-PARP and cleaved-caspase9, while downregulated the expression of pro-caspase3, and pro-caspase9 in CRC cells. The index of cleaved-PARP could be reversed by using Z-VAD-FMK, a classical apoptosis inhibitor in both SW480 and HCT116 cells, and we could also observe that the pro-caspase3 and pro-caspase9 were reversed in HCT116 cell (Fig. 4B and Supplementary Fig. 4B). Flow cytometry analysis also showed that with the addition of Z-VAD-FMK, the proportion of apoptotic cells significantly decreased (Fig. 4C,D). All the results suggested the occurrence of apoptosis in CRC cells during ATPP-PDT.

Fig. 4.

450-nm blue laser-mediated ATPP-PDT induces apoptosis in CRC cells. (A) Expression of apoptosis-related proteins in HCT116 and SW480 cells after 24 hours treatment in different treatment groups. (B) The impact of the apoptosis inhibitor Z-VAD-FMK on PDT-induced apoptosis was examined using western blotting. (C) Flow cytometry analysis of the effect of apoptosis inhibitor Z-VAD-FMK on ATPP-PDT induced apoptosis. (D) Quantitative analysis of (C) (n = 3, mean $\pm$ SD). **p 0.01, and ***p 0.001.

In order to further explore the potential mechanism of ATPP-PDT inducing cell apoptosis, we further made transcriptome RNA sequencing. As demonstrated in Fig. 5B, there were 3138 differentially expressed genes (DEGs), including 1531 upregulated genes and 1607 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the MAPK signal pathway, a classic pathway of apoptosis, was engaged in ATPP-PDT induced apoptosis (Fig. 5A). Phospho-p38 MAPK increased significantly in ATPP-PDT group in both HCT116 and SW480 cells (Fig. 5C and Supplementary Fig. 5A), with the addition of SB202190 (a selective p38 MAPK inhibitor), we could not only observe that the expression of phospho-p38 MAPK was down-regulated again, but found the process of cell apoptosis was inhibited (Fig. 5D and Supplementary Fig. 5B). All these results confirmed the vital role of p38 MAPK signal pathway in ATPP-PDT. What’s more, after adding ROS scavenger NAC, the p38 MAPK pathway was inhibited and the expression of apoptosis-related proteins was reversed (Fig. 5E and Supplementary Fig. 5C). Then, flow cytometry observed that both SB202190 and NAC could significantly decrease the proportion of apoptotic cells in HCT116 and SW480 cells (Fig. 5F–I and Supplementary Fig. 5D–G). These above results suggested that blue laser mediated ATPP-PDT induces apoptosis by ROS activating p38 MAPK signal pathway.

Fig. 5.

ATPP-PDT induces apoptosis by up-regulating ROS/p38 MAPK signal pathway. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differential gene expression (DEGs) in PDT group and ATPP-DTPA group in HCT116 cell. (B) Volcano plot of the DEGs in HCT116 cells between PDT group and ATPP-DTPA group. Western blotting analysis of the expression levels of p38 MAPK pathway-related proteins after the treatment of 24 h (C), and the effect of SB202190 (D) and NAC (E) on ATPP-PDT-induced apoptosis and the activation of p38 MAPK pathway. (F,G) Flow cytometry was used to analyze the changes of apoptosis of HCT116 cells after adding inhibitors SB202190 and NAC, respectively. Quantitative statistics were shown in the (H,I) respectively. (n = 3, mean $\pm$ SD). **p 0.01, and ***p 0.001.

To examined the in vivo therapeutic potential of ATPP-PDT, HCT116 cell subcutaneous xenograft models were constructed in BALB/c nude mice (Fig. 6A). On the 10th day after subcutaneous tumor implantation, the mice received caudal vein injection of ATPP-DTPA and irradiated the tumor site with 450-nm blue laser next day. Compared to other control groups, the tumor development of ATPP-PDT group was strongly inhibited, as seen by the significantly lower tumor volumes and weights (Fig. 6C–E) and the lack of significant changes in body weight (Fig. 6B). Next, we performed H&E and IHC of the tumor tissue and important organs from the mice (Fig. 6F and Supplementary Fig. 6B). In PDT group, substantially lower level of Ki-67 and higher expression of phospho-p38 MAPK and cleaved-PARP were discovered. Meanwhile, we didn’t observe any evident pathological alterations in the major organs, such as the heart, liver, spleen, lung and kidney (Supplementary Fig. 6A). All of the findings suggested that ATPP-PDT could successfully and safely stop CRC tumor development in vivo.

Fig. 6.

Effect of ATPP-PDT mediated by 450-nm blue laser in vivo. (A) The general schematic illustration of the in vivo experiment. (B,C) The body weight and tumor volume changes in mice across several treatment groups throughout the treatment. (D) Comparison of tumor weight after different treatment. (E) Visuals of the tumor tissues following various therapies. (F) Tumors from mice were stained with H&E and IHC analysis following various treatments. Scale bar = 50 µm and 20 µm. (n = 6, mean $\pm$ SD), ***p 0.001.