Pancreatic cancer is one of the most lethal malignancies in the world and is frequently diagnosed at advanced stages of disease; thus, it has a very poor 5-year survival rate (less than 5%) [
To date, clinical endoscopic ultrasound (EUS) is used in the first step to diagnose pancreatic diseases; and more recently, EUS-guided fine needle injection (EUS-FNI) of agents also has been used to treat pancreatic cancer or to control tumor-induced pain through nerve blockade [
The human pancreatic cancer cell line SW1990 was obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100
The research protocol was approved by the Ethics Committee of The Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). Athymic nu/nu male mice, aged 4–6 weeks and weighing 20–22 g, were obtained from the SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The animals were housed in a specific pathogen-free environment, where cages, bedding, food, and water were autoclaved for 1 week to adapt to the new surroundings before the animal experiments.
SW1990 cells at the exponential growth phase were harvested and resuspended in phosphate-buffered saline to a single cell suspension and then injected subcutaneously into the right flank of nude mice to establish the subcutaneous pancreatic cancer mouse xenograft model (1 × 106 cells per mouse). One week after tumor cell inoculation, the mice were randomized into six groups, with eight mice per group: (a) normal saline control, (b) 20% ethanol group, (c) 40% ethanol group, (d) 60% ethanol group, (e) 80% ethanol group, and (f) 95% ethanol group. The xenograft lesions of the mice were injected once with different concentrations of ethanol or saline using a 25-gauge needle at a single site.
To establish the animal model, mice were anesthetized through intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then a left lateral minilaparotomy was conducted by mobilizing the spleen to expose the pancreas. After that, 0.2 mL of SW1990 cells (1 × 107 cells/mL) was injected into the parenchyma of the pancreas, and the abdominal incision was sutured using a surgical staple. Growth of the pancreatic cancer xenografts was monitored by using a high-frequency EUS probe (GF-UCT240-AL5, Olympus Co. Ltd., Tokyo, Japan). Ten days after tumor cell injection, the mice were divided randomly into two groups with 10 mice per group: (a) control group with saline injection and (b) ethanol group with 80% ethanol injection. The intratumoral injections were guided by the percutaneous high-frequency EUS probe. Specifically, after sodium pentobarbital anesthesia, supine animals were placed on a board and the abdomen was soaked carefully with sterile deionized water. The ultrasonic images were obtained using the EUS probe with a water bag and the direct contact method. Under the EUS probe guidance, agents were injected slowly into the tumor xenografts of the mice. The dose of ethanol per injection was amended according to the regression equation of liver tumor ethanol ablation:
Growth of subcutaneous tumor xenografts was measured using a vernier caliper, while growth of the orthotopic tumor xenografts was measured by a high-frequency EUS probe at baseline and 7 days after treatment. The tumor volume (
After the mice were sacrificed, the tumor xenografts were excised, fixed in 10% buffered formalin, and embedded in paraffin for preparation of tissue sections (4
Blood samples were withdrawn from the tail vein at baseline as well as 24 h and 7 days after treatment. These blood samples were then assessed for serum amylase levels using an automatic biochemical analyzer (BioAssay Systems, Hayward, CA, USA).
All data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). The results were plotted as mean values ± standard deviation, and the data were evaluated by using one-way analysis of variance with the least-significant difference test for comparisons between groups. The data were considered statistically significant when
During the experiments, one mouse each in the 60% and 80% ethanol groups and two mice in the 95% ethanol group of the subcutaneous xenograft model died, which might have been due to the excessive ethanol dose. At 7 days after the single ethanol injection, tissue xenografts showed a large area of tumor necrosis in the mice injected with 60%, 80%, or 95% ethanol, whereas a very small area of tumor necrosis occurred in the mice injected with 20% or 40% ethanol. However, there was no necrosis present in the controls (Figure
Effect of ethanol injection on the subcutaneous pancreatic cancer cell mouse xenograft model. The tumor volume and relative tumor volume (RTV) of these mouse xenografts significantly changed after different doses of ethanol were injected into the tumor xenografts of nude mice ((a) 0%, (b) 20%, (c) 40%, (d) 60%, (e) 80%, and (f) 95%). (g) Summary of the data.
Histology of the subcutaneous pancreatic cancer cell mouse xenograft model after ethanol injection. On day 7, all mice were sacrificed and the tumor xenografts were resected for tissue processing and H&E staining. (a) 0%, (b) 20%, (c) 40%, (d) 60%, (e) 80%, and (f) 95%. The original magnification was 100x.
During the experiments, two mice died at 24 h and 72 h after the ethanol injection. The lethal rate was 20% in the experimental group. Immediately after the injection of 80% ethanol, a hyperechoic area was noted in the tumor xenografts (Figure
EUS-FNI of ethanol ablation of the orthotopic human pancreatic cancer cell mouse xenografts. (a) Preinjection. To ablate the orthotopic pancreatic cancer cell mouse xenograft, a fine needle was inserted into the xenograft lesion. (b) Postinjection. 80% ethanol was injected into the xenograft. Immediately after the injection of 80% ethanol, a hyperechoic area was noted in the tumor lesion.
Effects of ethanol injection on the control of orthotopic pancreatic cancer cell mouse xenografts. (a) Tumor volume on day 0 after ethanol injection. (b) Tumor volume on day 7 after ethanol injection. (c) Tumor volume on day 0 after saline injection. (d) Tumor volume on day 7 after saline injection. (e) Summary of the data.
Furthermore, representative H&E-stained tumor xenograft sections showed that on day 7 after the ethanol injection, the tumor xenografts had a large area of necrosis in the 80% ethanol group, but there was no necrotic or damaged region in the control group (Figure
Histology of the orthotopic pancreatic cancer cell mouse xenograft model after ethanol injection. On day 7, all mice were sacrificed and the tumor xenografts were resected for tissue processing and H&E staining. (a) Saline injection. (b) 80% ethanol injection. Severe coagulation and necrosis occurred in the tumor xenografts after the 80% ethanol injection.
The serum amylase level was just slightly elevated in mice at 24 h after the 80% ethanol injection and returned to the baseline level on day 7 after treatment (Figure
Change in serum amylase levels after ethanol injection into the orthotopic pancreatic cancer cell mouse xenografts. Blood samples were collected from the mouse-tail vein on days 0, 1, and 7 after ethanol injection and then assessed for serum amylase levels.
To date, treatment of advanced pancreatic cancer with chemoradiation therapy is usually ineffective, and novel strategies are urgently needed to control this deadly disease and to improve the survival of patients. In the current study, we established an orthotopic pancreatic cancer mouse xenograft model and then assessed the effectiveness of high-frequency EUS-FNI of ethanol on ablation of pancreatic cancer cell xenografts. Our data showed that such treatment with ethanol could be a useful strategy to control pancreatic cancer. It is true that EUS has been used in clinical practice for more than 30 years since it was first developed for pancreatic disease diagnosis and staging of pancreatic malignancies. Only more recently, EUS has evolved into a useful therapeutic tool for pancreatic tumors. EUS-FNI, a successful minimally invasive approach, has been introduced as a novel technique for the local delivery of antitumor agents, including ethanol, brachytherapy, and ONYX-105 [
In the current study, we demonstrated the ethanol antitumor activity in the subcutaneous and orthotopic pancreatic cancer cell mouse xenograft models. We found that ethanol (20–95%) treatment resulted in local tumor necrosis in the subcutaneous model and that the antitumor activities of the 80% and 95% ethanol groups were much better than that of the 60% ethanol group, while there was no difference between the 80% and 95% ethanol groups. Matthes et al
However, there are possible risks of ethanol injection in the treatment of the orthotopic pancreatic cancer cell mouse xenograft model compared to that of the subcutaneous pancreatic cancer cell mouse xenograft model. Ethanol may induce pancreatitis to a certain degree and injure the surrounding tissues, which are more difficult to manage than in the subcutaneous model. Our current data showed that the serum amylase level was just slightly elevated in mice at 24 h after 80% ethanol injection and returned to the baseline level on day 7 after treatment. In addition, 80% ethanol did induce local complications, such as necrosis in the liver and spleen. In other studies, Gan et al
In conclusion, our current study suggests that EUS-FNI of ethanol into pancreatic cancer cell mouse xenografts is feasible, safe, and effective and that the 80% ethanol injection reduces the volume of pancreatic cancer xenografts in the orthotopic pancreatic cancer mouse model. Further studies will assess multiple injections and the time required to treat pancreatic cancer in more detailed assessments of its safety and efficacy.
The authors declare that there are no conflicts of interest in this work.
Wen-Ying Zhang and Zhen-Dong Jin contributed equally to this work.
This study was supported in part by a grant from the Xinhua Group Foundation, Shanghai, China (#13xj22013).