Grossly, the tumors were presented subcutaneously at the transplanted areas of all xenograft nude mice in each group. In the xenograft tumors, ulcerated and necrosis were found in several nude mice of each experimental group (Supplementary data Fig. 1). In other internal organs, there was no significant change in gross examination except mild to moderate pulmonary congestion. The metastasis of tumor cells in both intrathoracic and intraabdominal organs was not macroscopically observed. Besides, we also did not observe a significant change in both hematology and blood chemistry profiles among different experimental groups.

The average tumor volume (mm³) was calculated on day 0 (initial day) and day 30 (final day) of the experiment. There was no significant difference (p > 0.05) between the average volume of the xenograft tumors over the course of treatment. Furthermore, there was no statistical difference (p > 0.05) in the percentage of tumor volume change among groups (Supplementary data Fig. 2).

To evaluate the histopathological morphology of the transplanted tumors on the xenograft nude mice, the H&E–stained section of an individual xenograft tumor was examined and evaluated under a light microscope (40×). The xenograft tumors were arranged into the glandular structure with fibrovascular stroma surrounding. Neoplastic cells were round to oval, lobulated with the intralesional necrotic area, and surrounded by clearly defined borders. The necrotic areas were frequently represented on the edge of xenograft tumors in the treatment and PC groups, while occasionally found in the center of the xenograft tumor in the NC group. The transplanted tumors displayed similar characteristics to the original neoplasm which was pancreatic ductal adenocarcinoma. In subtype classification, all the examined tumors mirrored the characteristics of adenosquamous carcinoma according to the World Health Organization (WHO) classification (Supplementary data Fig. 3). The metastasis of tumor cells in both intrathoracic and intraabdominal organs was not microscopically observed. Besides, there was no significant change in the collected organs, except mild pulmonary congestion (Supplementary data Fig. 4). Moreover, this study performed immunohistochemistry (IHC) to detect the proliferation of blood vessels in the xenograft tumors via a specific antibody (CD31/PECAM-1). The immunoexpression of the CD31/PECAM-1 antibody showed no significant proliferation (p > 0.05) of the blood vessels in the xenograft tumors among groups (Supplementary data Fig. 5).

In the area of the predominated number of apoptotic cells, the number of mitotic cells was rare (Fig. 1a). For mitotic cell count, the number of mitotic cells (cells/high–power field (HPF)) was highest in the negative control (NC) group compared with those in cannabinoid–treated groups and the positive control (PC) group. The difference in the mitotic cell count between the NC group and experimental groups was statistically significant (p < 0.01) (Fig. 1b). In contrast, the apoptotic cell count (cells/HPF) was significantly increased (p < 0.001) in cannabinoid–treated groups, and the PC group relative to the NC group (Fig. 1c). When comparing the mitotic/apoptotic (M/A) ratio, there was dramatically decreased (p < 0.001) in the PC group and cannabinoid–treated groups compared to the NC group (Fig. 1d).Figure 1

Effect of cannabinoids on the mitotic and apoptotic index. (a) The xenograft tumors performed scattered mitotic cells (arrow) and apoptotic cells (arrowhead) (H&E stained, 40×, scale bar = 50 µm). (b–d) The mitotic (b), apoptotic cell count (cells/HPF) (c), and M/A ratio (d) among the NC group, PC group, and cannabinoids–treated groups at a dose of 1, 5, and 10 mg/kg BW. The values were presented as mean ± SD. ***p < 0.01; ****p < 0.001 (one–way ANOVA and post hoc test).

To investigate the effect of cannabinoids on the human PDAC xenograft tumors through the mechanism of tumor cell death, the mRNA expression level of apoptotic–related genes including BAX, BCL-2, Caspase-3, and Caspase-8 was performed. As demonstrated in the results, the relative expression level of the pro–apoptotic BAX gene was significantly different (p < 0.001) in the PC and cannabinoid-treated groups at a dose of 5 and 10 mg/kg body weight (BW), whereas there was no statistical difference (p > 0.05) in cannabinoid–treated group at a dose of 1 mg/kg BW relative to the NC group (Fig. 2a). The relative expression level of the anti-apoptotic BCL-2 gene was dramatically lower (p < 0.001) in the PC and cannabinoid–treated groups compared to the NC group (Fig. 2b). The BAX/BCL-2 relative expression ratio significantly rises (p < 0.001) in the PC and cannabinoid-treated groups at a dose of 5 and 10 mg/kg BW relative to the NC group (Fig. 2c). However, the relative expression level of the BAX/BCL-2 ratio in cannabinoid-treated group at a dose of 1 mg/kg BW was not significantly different (p > 0.05) in comparison to the NC group (Fig. 2c). The relative expression level of the pro-apoptotic Caspase-3 gene dramatically increased (p < 0.05) in the PC and cannabinoid–treated groups relative to the NC group (Fig. 2d). Moreover, the relative expression level of the pro–apoptotic Caspase-8 gene was not significantly different (p > 0.05) among PC, cannabinoid–treated groups, and the NC group (Fig. 2e).Figure 2

The mRNA expression levels of BAX gene (a), BCL-2 gene (b), BAX/BCL-2 ratio (c), Caspase-3 gene (d), and Caspase-8 gene (e) among the NC group, PC group, and cannabinoids-treated groups at a dose of 1, 5, and 10 mg/kg BW. The values were presented as mean ± SD. **p < 0.05; ****p < 0.001; ^nsp > 0.05 (Kruskal–Wallis test).

Correlation with the aim to investigate the effect of cannabinoids on the human PDAC xenograft tumors via the mechanism of tumor cell death, IHC was carried out to detect the expression of proliferative markers (Ki-67 and PCNA), and apoptotic–related markers (BAX, BCL-2, Caspase-3, Caspase-8, and p53) (Fig. 3a,d). As presented in the results, the percentage of Ki-67-positive labeling cells was significantly reduced (p < 0.05) in cannabinoid–treated group at a dose of 5, 10 mg/kg BW, and the PC group, meanwhile there was not statistically different (p > 0.05) in cannabinoid–treated group at a dose of 1 mg/kg BW relative to the NC group (Fig. 3b). However, the percentage of PCNA–positive labeling cells was dramatically different (p < 0.01) among cannabinoid–treated groups and the PC group when compared with the NC group (Fig. 3c). On the other hand, consistent with the gene expression results, cannabinoids and the PC groups significantly increased (p < 0.05) the expression of BAX protein, BAX/BCL-2 ratio, meanwhile statistically decreased (p < 0.001) the expression of BCL–2 protein and Caspase-3 protein (Fig. 3e–h). However, the expression of BAX protein and BAX/BCL-2 ratio was not different (p > 0.05) between cannabinoid–treated group at a dose of 1 mg/kg BW and the NC group (Fig. 3e,g). Furthermore, there was no positive labeling of Caspase-8 protein in all groups (Fig. 3d). For p53 protein expression, the percentage of positive labeling cells was significantly reduced (p < 0.01) in the PC group, meanwhile there was not statistically different (p > 0.05) in cannabinoid–treated groups compared to the NC group (Fig. 3i).Figure 3

The immunoexpression of anti-proliferative markers (Ki-67 and PCNA) (a), and anti–apoptotic markers (BAX, BCL-2, Caspase-3, Caspase-8, and p53) (d) among the NC group, PC group, and cannabinoids–treated groups at a dose of 1, 5, and 10 mg/kg BW. The intranuclear labeling of Ki–67, PCNA, Caspase-3, and p53; intracytoplasmic labeling of BAX, BCL-2 in the xenograft tumors were displayed as brown color (DAB labeled, Mayer’s hematoxylin counterstained, 40×, scale bar = 50 µm). The expression levels of protein Ki-67 (b), PCNA (c), BAX (e), BCL–2 (f), BAX/BCL-2 ratio (g), Caspase-3 (h), and p53 (i) among the NC group, PC group, and cannabinoids–treated groups at a dose of 1, 5, and 10 mg/kg BW. The values were presented as mean ± SD. **p < 0.05; ***p < 0.01; ****p < 0.001 (Kruskal–Wallis test).

The Western blot (WB) was conducted to confirm the gene and IHC expressions. These results showed that the relative protein expression levels of BAX, BAX/BCL-2 ratio, and Caspase-3 increased significantly (p < 0.05 or p < 0.001) in cannabinoid–treated groups and the PC group relative to the NC group (Fig. 4b, d, and e). Contradictorily, the relative protein expression levels of BAX in cannabinoid-treated group at a dose of 1 mg/kg BW were not significantly different (p > 0.05) compared to the NC group (Fig. 4b). Meanwhile, the relative protein expression levels of BCL–2 were dramatically decreased (p < 0.001) in cannabinoid-treated groups compared to the NC group (Fig. 4c). Besides, the Caspase-8 protein expression levels were dramatically increased (p < 0.001) in the PC group when compared with the NC group, meanwhile, there was not statistically significant (p > 0.05) in cannabinoid-treated groups and the NC group (Fig. 4f).Figure 4

The representative WB results (a) and relative expression levels of protein BAX (b), BCL–2 (c), BAX/BCL–2 ratio (d), Caspase-3 (e), and Caspase-8 (f) among the NC group, PC group, and cannabinoids–treated groups at a dose of 1, 5, and 10 mg/kg BW. The values were presented as mean ± SD. **p < 0.05; ****p < 0.001 (Kruskal–Wallis test).

The cannabinoid solution with a ratio of THC:CBD (1:6) was used in this study according to the formula of the Government Pharmaceutical Organization, Thailand. The present study used the ratio of THC:CBD (1:6) followed by the previous publication⁵³.

All human PDAC xenograft tumors of this study were recruited from the prior study⁵³. The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of The National Cancer Institute, Thailand (Approval No.272_2019RB_IN602) and Lerdsin Hospital, Department of Medical Services (Approval No.ACE–F–v03–02). All animal experiment procedures were completely followed according to the approved guidelines and ARRIVE guidelines. Briefly, 25 male immunodeficient mice (nude mouse, BALB/cAJcl–nu) at 4-weeks-old were acclimatized for one week at a strictly hygienic conventional laboratory system. All mice were subcutaneously injected with 5 × 10⁶ human PDAC (Capan–2, HBT–80) cells at the right flank region under aseptic conditions. The xenograft tumors were measured once every 3 days by a vernier caliper. Tumor volume was calculated according to the described formula: Volume (mm³) = (Length x Width²)⁵⁴. The treatments were started during the tumor volume developed around 200 mm³. Five mice were randomly arranged into a treatment group according to the completely randomized design. Two controls and three experimental treatments were included (1) NC group: mice were gavaged with sesame oil; (2) PC group: mice were intraperitoneally injected with 5–FU at a dose of 20 mg/kg BW, 3 times per week; (3), (4), (5) experimental group 1, 2, 3: mice were gavaged with cannabinoid solution at a dose of 1, 5 and 10 mg/kg BW/day, respectively. The experimental scheme is presented in Fig. 5.Figure 5

The experimental scheme of this study.

The xenograft tumors and internal organs (lung, heart, liver, pancreas, spleen, and kidney) of experimental nude mice were collected and fixed with 10% neutral buffered formalin for 24 h subsequent to euthanasia, necropsy, and gross examination. In all collected samples, routine tissue processing was performed including paraffin embedding and H&E stain. The morphological evaluation and subtype classification of PDAC were accomplished according to the previous publication of the WHO⁵⁵.

The number of mitotic and apoptotic cells in individual tumor sections was counted as described in the previous study⁵⁶. For the cell count, a total of ten areas were randomly captured at an HPF (40x, area equals 2.37 mm²) per section. Mitotic and apoptotic cell counts were accomplished by manually counting in each group. The average number of mitotic and apoptotic cells per section was followed by the formula of the number of expressed cells per HPF (cells/HPF). The mitotic/apoptotic (M/A) ratio was calculated via dividing the mitotic index by the apoptotic index. A double–blind study was performed to evaluate the results by certified veterinary pathologists.

The innuPREP RNA Mini Kit 2.0 (Analytik Jena AG, Jena, Germany) was selected to extract the total RNA from fresh xenograft tumors following the manufacturers’ instructions. The aqueous phase of the final mixture was collected and kept at − 80 °C until used for further processing. The synthesis of cDNA was transfected by using the qPCRBIO cDNA Synthesis Kit (PCR Biosystems, London, United Kingdom). The synthesized process followed the manufacturers’ instructions. The concentration of total RNA and synthesized products was checked by the NanoDrop Lite spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA).

The expression of apoptotic–related genes was amplified using the gene–specific primer pairs (Table 1) as focusing the previous publications^57–60. qRT–PCR was performed on a Rotor–Gene Q thermocycler (Qiagen, Hilden, Germany). Briefly, 20 ng of cDNA were well mixed in 10 µL of 2 × qPCRBIO SyGreen Mix (PCR Biosystems, London, United Kingdom), 400 nM of individual primer, and adjusted into a total of 20 µL final volume, according to the manufacturers’ instructions. The thermal cycle of the qRT–PCR reaction consisted of polymerase activation at 95 °C for 2 min, following 40 cycles of denaturation at 95 °C for 5 s, and extension for 30 s at 60 °C. qRT–PCR determined the cycle threshold (Ct) value of individual target genes. An individual sample was carried out in duplicate per each target gene. The relative quantification among target genes was accomplished through the earlier 2^–ΔΔCt method⁶¹. β–actin gene was chosen as an internal control following the prior descriptions^62,63. The expression level of the individual target genes among experimental groups was averaged and displayed as a normalized ratio. The BAX/BCL–2 ratio was calculated by dividing the normalized ratio of BAX and BCL–2 gene expression.Table 1

The sequences of qRT–PCR primer pairs used in the current study.

Raffin–embedded tissues were deparaffinized with xylenes and rehydrated with a gradual decrease in alcohol concentration. The slides were pretreated for antigen retrieval, conditions following the antibody (Table 2). For blocking non–specific antibodies, all specimens were blocked with 1% bovine serum albumin for 30 min at 37 °C, subsequently incubated with primary antibody at 4 °C, overnight (Table 2). The secondary antibody was following applied, using a biotinylated goat anti–mouse/anti–rabbit antibody Envision (Dako, Glostrup, Denmark) for 1 h at RT. A freshly prepared 3, 3′–diaminobenzidine (DAB) solution (Dako, Glostrup, Denmark) was added to visualize the colored reaction. Finally, the slides were immersed in distilled water, counterstained with Meyer’s hematoxylin stain, and permanently mounted. The negative control was experimental specimens, following the same procedure, except for incubating with a primary antibody. A double–blind study was performed to evaluate the immunoexpression results by certified veterinary pathologists.Table 2

The immunohistochemical primary antibody, working dilutions, antigen retrieval, and positive staining pattern.

In individual immunoexpression specimen, a total of ten areas were randomly captured by the digital imaging system at an HPF (40×, area equals 2.37 mm²). In a group of proliferative markers (Ki–67 and PCNA), p53, and Caspase-3, the number of positive cells per total nucleated cells was evaluated. The data were calculated and displayed as a percentage (%) of positive cells per total nucleated cells. On the other hand, the percentage of positive cells per 1 mm² (%IHC positive area/mm²) within a group of related–apoptotic markers (BAX and BCL–2) were calculated by the image analysis program (NIS–Elements Analysis D, Nikon, Japan). The BAX/BCL–2 ratio was calculated by dividing the normalized ratio of BAX and BCL–2 protein expression.

Western blot analysis was conducted with the extracted protein from the xenograft tumors following the guideline of Minute Total Protein Extraction Kit for Animal Cultured Cells and Tissues (Invent Biotechnologies, Massachusetts, USA) and stored at − 20 °C. The lysed protein was loaded onto the 10% SDS–PAGE polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Santa Cruz Biotechnology, Texas, USA). Thereafter, the membrane was blocked with 5% skim milk for 1 h at RT and was incubated overnight with primary antibody (Table 3) at 4 °C. Following, anti–mouse IgGk BP–HRP (Cat. Num. sc–516102, Santa Cruz Biotechnology, Oregon, USA) or anti–rabbit IgG–HRP (Cat. Num. sc–2357, Santa Cruz Biotechnology, Oregon, USA) at 1:5,000 dilution was developed as secondary antibody for 1 h at RT. For visualization, the LumiFlash Prime Chemiluminescent Substrate (Energenesis Biomedical, Taipei, Taiwan) and ChemiDoc and ChemiDoc MP Imaging Systems (Bio–Rad, California, USA) were chosen to detect and scan the results, respectively. The labeling intensity of samples was evaluated via the signal strength, using ImageJ software⁶⁴.Table 3

The primary antibodies used for WB in the study.

The one–way analysis of variance (ANOVA) and post hoc test were considered to test the difference in the quantitative variables (apoptotic index; mitotic index; Ki–67, PCNA, p53, and Caspase-3 protein expression) as well as semi–quantitative variables (BAX and BCL–2 protein expression) among groups. The qRT–PCR and western blot results were evaluated by a nonparametric test to compare the differences between the experimental groups, using the Kruskal–Wallis test. All statistical analyses were performed by the SAS software version 9 for Windows (SAS Institute, Inc., North Carolina, USA). The values were presented as mean ± standard deviation (SD). P values below 0.05 were considered the criterion for statistically significant.

Supplementary Figure 1.