This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.
Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of metastasis that accompanies unresectable gastric cancer and is a definitive determinant of prognosis. Preventing and controlling the development of peritoneal metastasis could play a role in helping to prolong the survival of gastric cancer patients. A non-invasive and efficient imaging technique will help us to identify the invasion and metastasis process of peritoneal metastasis and to monitor the changes in tumor nodules in response to treatments. This will enable us to obtain an accurate description of the development process and molecular mechanisms of gastric cancer. We have recently described experiment using dual energy CT (DECT) and positron emission tomography/computed tomography (PET/CT) platforms for the detection and monitoring of gastric tumor metastasis in nude mice models. We have shown that weekly continuous monitoring with DECT and PET/CT can identify dynamic changes in peritoneal metastasis. The sFRP1-overexpression in gastric cancer mice models showed positive radiological performance, a higher FDG uptake and increasing enhancement, and the SUVmax (standardized uptake value) of nodules demonstrated an obvious alteration trend in response to targeted therapy of TGF-β1 inhibitor. In this article, we described the detailed non-invasive imaging procedures to conduct more complex research on gastric cancer peritoneal metastasis using animal models and provided representative imaging results. The use of non-invasive imaging techniques should enable us to better understand the mechanisms of tumorigenesis, monitor tumor growth, and evaluate the effect of therapeutic interventions for gastric cancer.
Gastric cancer (GC) remains the fourth most common malignancy and the second-leading cause of cancer mortality worldwide1. Although the accuracy in diagnosis and treatment of gastric cancer has been greatly improved, peritoneal metastasis is the most key point of gastric cancer prognosis or recurrence and is a definitive determinant of postoperative death2. It is generally accepted that peritoneal dissemination is a life-threatening mode of metastasis, wherein the disease becomes uncontrollable and the prognosis of the patient is poor once peritoneal dissemination is established. Therefore, the detection and therapeutic effect evaluation of gastric cancer peritoneal metastasis is crucial for clinical practice.
The increasing incidence and mortality of gastric cancer had spurred researchers to identify its molecular mechanisms. The high expression of genes such as secreted frizzled-related protein 1 (sFRP1) might lead to activation of the signal pathway in the early stages of gastric cancer, promoting the process of tumor growth, proliferation, differentiation, and apoptosis3,4,5,6,7. sFRP1-overexpression cells showed an increase in the expression of TGFβ, its downstream targets, and TGFβ-mediated EMT8. Previous studies have demonstrated that the TGF-β1 level is correlated with peritoneal metastasis and the TNM stages of gastric cancer. We have described the changes in cancer cell proliferation regulated by sFRP1 overexpression and TGF-β1 inhibition, and established animal models for peritoneal metastasis to show the performance of tumor imaging under the effects of gene regulation.
Animal models for gastric cancer are indispensable tools for researching tumor development and experimenting with various therapeutic strategies without having to sacrifice animals. Animal models have proven useful in studying the formation mechanisms of tumors and cells of origin, determining the presence of cancer stem cells, and examining various novel therapeutic strategies. Therefore, a real-time non-invasive technique can provide an accurate description of the development of gastric tumors and tumor response to treatments, which can identify the development of peritoneal metastasis nodules in nude mice and monitor the changes of a tumor in response to various experimental and therapeutic interventions.
Currently, multi-detector CT (MDCT) plays an important role in the TNM staging of gastric cancers and is useful for predicting tumor resectability preoperatively9. However, radiological studies of patients with histologically proven gastric carcinoma have mainly been based on morphology. DECT imaging extends the parameters to reflect functional information by providing monochromatic images and may be helpful for improving the N staging accuracy for gastric cancers. Furthermore, this technique will enable the acquisition of material-decomposition images, which may be useful to differentiate between differentiated and undifferentiated gastric carcinoma, and between metastatic and non-metastatic lymph nodes10. With the introduction of DECT, the functional imaging aspect of CT has also been added to clinical applications, contributing to evaluations of therapeutic efficacy and predicting patient prognoses11,12,13. PET/CT is a useful imaging technique for the detection and staging of gastric cancer and can evaluate the recurrence of the tumor effectively14. Tumor cell proliferation and angiogenesis were both considered to be necessary in the development of a detectable tumor15, tumor nodules showed a positive performance with higher SUVmax on PET/CT. Based on their preference for aerobic glycolysis, 18F-FDG, a glucose analog, has been exploited as a promising tracer in the diagnosis of malignancies, combined with PET/CT16. This method relies on the rapid glucose consumption of tumor tissue and has broad clinical applications, including assisting in the detection, staging, and evaluation of the prognosis of tumors, as well as monitoring the tumors' response to therapy17,18. As non-invasive methods, DECT and PET/CT have been utilized to diagnose malignant tumors and to assess tumor response to various therapies.
Our group has been using this non-invasive imaging method with DECT and PET/CT scanners to detect and monitor the process of tumor growth and metastasis in living mice19. We explored imaging findings induced by the sFRP1-overexpression in gastric cancer cells in vivo using nude mice, with DECT and PET/CT, and described the changes of the SUVmax value following targeted therapy by the TGF-β1 inhibitor to confirm the development of tumor nodules in the peritoneum after gene induction, and also studied the changes in tumor nodules in response to experimental treatments. In this paper, we present detailed procedures for modeling gastric tumor peritoneal metastasis in mice, and its detection and monitoring with DECT and PET/CT.
This work was performed in strict accordance with the standards established by the Guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University and was approved by the laboratory Animal Ethics Committee of Ruijin Hospital.
1. Gastric Cancer Peritoneal Metastasis Animal Model
2. DECT for Peritoneal Metastasis Animal Model
NOTE: The animal imaging experiment was achieved on the dual energy CT scanner (see Table of Materials). We created the related DECT imaging protocol according to the previous studies.
3. PET/CT for Peritoneal Metastasis Animal Model
NOTE: See the table of materials for the PET/CT imager used. We created the related PET/CT imaging protocol according to this article21.
DECT and PET/CT scanning were performed on nude mice after two weeks of cell line injections. GSI images yielded excellent results for displaying subcutaneous metastasis beyond the contour of the abdomen for the sFRP1 overexpression group, and metastasis with peripheral enhancement was confirmed by color-scale image (Figure 1a-c). PET/CT images depicted focally abnormal FDG uptake of metastasis, including in the peritoneal and subcutaneous metastases (Figure 1d). The peritoneal metastasis and large subcutaneous metastasis shown on the DECT and PET/CT images were further illustrated by gross specimen and histological section (Figure 1e-f). Compared with the positive expression group, there were no visible lesions, obvious abnormal enhancements, or high FDG uptake in the abdominal cavity in the sFRP1 empty loading group from DECT and PET/CT images (Figure 2a-b). Though the images of the gross specimen and histological results confirmed the successful implantation for this group (Figure 2c-d).
The intervention treatment of TGF-β1 inhibitor and placebowere performed on nude mice after two weeks of cell line injections, and DECT and PET-CT scanning were performed on nude mice after two weeks of treatment. To confirm the process of the formation of peritoneal metastasis nodules in mice from the initiation of target therapy, follow-up DECT and PET/CT scans were performed. Non-invasive imaging scans were performed to assess the effect of TGF-β1 targeted treatment. The images for the mice in the TGF-β1 treatment group depicted obvious enhancement and focal abnormal FDG uptake of metastases in the coronal fused images of DECT and PET/CT (Figure 3a-b). Gross specimens illustrated only 8 nodules of peritoneal metastases (Figure 3c) with diffused distribution in the abdominal cavity. Quantitatively, Figure 3 showed moderate peripheral enhancement on DECT and reduced FDG uptake, with an SUVmax close to 0.83. On the other hand, mice in the control group that were given normal saline also showed visible lesions and focally abnormal uptake of metastases in the coronal fused images of DECT and PET/CT (Figure 4a-b). Gross specimens illustrated 22 nodules of peritoneal metastases (Figure 4c), and the local metastasis nodules were adherent to the abdominal cavity. The SUVmax values in the tumors were not changed (at 1.26) for mice in the control group that were given normal saline.
It is noteworthy that sometimes the intestinal tract will cause FDG mild ingestion and the bright region in the images will produce false positive results.The heart and bladder will also gather a lot of FDG, which may show as a bright spot in the images.It is necessary to avoid the relevant level images to determine the real FDG uptake area of tumor.
Figure 1: Tomogram of sFRP1 overexpression group peritoneal metastasis model from DECT, PET/CT and corresponding HE staining. (a–c) GSI monochromatic images in portal phase: (a) transverse monochromatic image, (b) transverse color-fused image, (c) coronal color-scale image; (d) coronal fused images of PET/CT, (e) gross specimen, (f) histological section. Arrows indicate the peritoneal metastatic nodules, while arrow heads indicate the subcutaneous metastasis. Figure 1f is the pathological staining result of a tumor nodule; the image shows the morphology and distribution of tumor cells; Scale bar = 100 µm. This figure has been modified from reference19. Please click here to view a larger version of this figure.
Figure 2: Tomogram of sFRP1 empty loading group peritoneal metastasis model from DECT, PET/CT and histological analysis. (a) GSI color-scale fused images obtained in portal phase, (b) fused PET/CT image, (c) gross specimen, and (d) histological section. No visible lesion, obvious abnormal enhancement or high FDG uptake was shown in the abdominal cavity. (c) and (d) confirmed the successful implantation. The arrow heads in Figure 2c pointed out the peritoneal metastatic nodules caused by the SGC-7901/vector cell lines. The heart and bladder showed obvious FDG elevation in Figure 2b. Figure 2d is the pathological staining result of a tumor nodule; the image shows the morphology and distribution of tumor cells; Scale bar = 100 µm. This figure has been modified from reference19. Please click here to view a larger version of this figure.
Figure 3: Tomogram of TGF-β1 treatment group peritoneal metastasis model by DECT and PET/CT and corresponding gross specimen. (a) the coronal fused image of DECT, (b) the fused images of PET/CT. (c) gross specimen. The heart and bladder showing obvious FDG elevation in the Figure 3b. Gross specimen illustrated 8 nodules of peritoneal metastases. Arrows pointed out the metastatic nodules corresponding to the radiographic finding. Please click here to view a larger version of this figure.
Figure 4: Tomogram of TGF-β1 control group peritoneal metastasis model by DECT and PET/CT and corresponding gross specimen. (a) The coronal fused image of DECT, (b) the fused images of PET/CT, and (c) gross specimen. Gross specimen illustrated 22 nodules of peritoneal metastases. Arrows pointed out the metastatic nodules corresponding to the radiographic finding. Please click here to view a larger version of this figure.
Supplementary Figure 1: Sample transverse color-fused image. Please click here to view a larger version of this figure.
Supplementary Figure 2: Sample coronal color-fused image. Please click here to view a larger version of this figure.
Animal models have been used widely in the study of molecular mechanisms underlying gastric cancer, and to experiment with various therapeutic strategies23,24,25. In this study, we have described a detailed protocol for gastric cancer peritoneal metastasis nude mice modeling, using DECT and PET/CT to image gastric tumors for identifying tumor cell proliferation in real-time, and monitoring peritoneal metastasis and responses to therapeutic interventions in gastric cancer animal models. This method could enable researchers who are involved in studying the molecular mechanisms of gastric cancer, or experiments imaging tumors, to establish more integrated and precise plans. In addition, we described the use of DECT and PET/CT devices that can serve as platforms for discovering tumor imaging performance with gene regulation and target therapy. Therefore, the method may be used by scientists to understand and explore the biological processes of cancer recurrence and progression. We have demonstrated that the non-invasive imaging modality could detect increased tumorigenesis by the overexpression of genes with positive results on DECT and PET/CT. Simultaneously, peritoneal metastasis after therapeutic intervention with targeted inhibitor showed negative FDG uptake performance on PET/CT. The SUVmax of tumor nodules presented a downward trend by extension of the treatment cycle.
In our study, we used the change in FDG uptake as an assessment indicator of therapeutic effects for peritoneal metastasis. Great effort has been put forth to show that FDG uptake is associated with tumor aggressiveness26. Progressive gastric carcinomas, represented by the depth of invasion, lymphatic permeation, vascular invasion, and tumor size, show higher FDG uptake27. In terms of quantitative evaluation, studies suggested that the SUVmax has a positive correlation with proliferation in various malignancies15,28. Our results demonstrated that compared with the control group, sFRP1 overexpression positively induced visibly larger nodules with significantly increasing enhancement and higher FDG uptake in peritoneal tumors, as was evidenced in Figure 1. In addition, the SUVmax showed an obvious alteration trend in the target treated groups, in contrast to no change in the control group. These results were demonstrated in Figure 3 with a decreased FDG uptake, with an SUVmax close to 0.83 for the treatment group, as opposed to an unchanged SUVmax value of close to 1.26 for mice in the control group that were given normal saline (Figure 4). Our results indicated that non-invasive imaging techniques, such as DECT and PET/CT, provide the possibility of using image technology to assess information at the molecular level in tumor cells and demonstrated the validity of combining the applications of DECT and PET/CT to provide a viable, reproducible, and non-invasive imaging strategy to monitor tumor nodules induced by gene modulation for gastric cancer research. Since the FDG uptake is associated with tumor aggressiveness26, the utilization of PET/CT imaging to assess the degree of tumor invasion and treatment is feasible.
In our previous studies, we have found that the injection time and method could influence the imaging results of DECT scanning. As the circulation chanages quickly in mice, and the enhancement of peritoneal metastasis mainly occurs in the arterial phase, the peritoneal metastases nodules may not be displayed completely if the scan begins significantly later than the injection time. The nude mice should be placed in a warm and clean environment after the examination, letting the mice rest for 12 h before PET/CT imaging to avoid the effects on the nodules' uptake of 18F-FDG in PET/CT imaging with excessive residual contrast agent in the body. Attention should be paid to the injection time and activity in the preparation and injection of FDG. The optimal activity concentration of 18F-FDG for PET/CT was about 100-200µCi/100µL for each injection. Too high of a concentration could increase the burden of circulatory system and result in the death of mice, while too low of a concentration might interfere with tumor uptake imaging. So, it is critical to ensure the efficiency and accuracy of the 18F-FDG configuration. Make sure that the PET/CT imaging position of nude mice is consistent with that of the DECT imaging to facilitate the matching of tumor images.
There are several limitations to our study. The limited resolution of DECT may contribute to the negative performance for visibility as some peritoneal tumors may exhibit insufficiently increasing size. It has been known that PET/CT has low specificity and a lack of anatomical localization, and the apoptosis and necrosis of tumor cells induced by chemotherapeutic drug interventions could influence the 18F-FDG uptake29,30. In addition, the normal physiological activity in bowel loops and 18F-FDG retention in ureters and bladder can contribute to false positives emerging in PET/CT images31. The process of peritoneal metastasis in nude mice models is difficult to detect and monitor, so the choice of appropriate time in therapeutic intervention and imaging experiments is particularly important. Therefore, the early diagnosis of small tumors is still a problem to be solved.
In conclusion, we have described a method that exploits DECT and PET/CT imaging technology for the accurate detection and evaluation of the efficacy of targeted therapy. Our results demonstrate that non-invasive imaging using the described protocols allows for the monitoring and evaluation of peritoneal metastasis progression using animal models. Applications of this method will be easily adapted for preclinical research aiming to discover gastric cancer peritoneal metastases, which may be useful to evaluate the disease diagnostic or therapeutic modalities.
The authors have nothing to disclose.
This work was supported by the NSFC (No. U1532107) and Shanghai Jiao Tong University Biomedical Engineering project (No. YG2014MS53). The authors would like to acknowledge Jianying Li and Yan Shen for their helpful comments and technical support efforts in developing the DECT and PET/CT imaging method.
Iohexol | BEJING BEILU PHARMACEUTICAL CO,LTD | NMPN:H20053800 | non-ionic contrast medium for DECT scan |
normal saline | HUNAN KELUN PHARMACEUTICAL CO,LTD | NMPN:H43020455 | placebo of control group |
BALB/c nude mice | SLAC LABORATORY ANIMAL | BALB/cASlac-nu | animal model |
SGC-7901 cells | Library of typical culture of Chinese academy of sciences | TCHu 46 | gastric cancer cell |
SB431542 | Selleck | No.S1067 | TGF-β1 inhibitor |
GE Discovery CT750 HD | GE Healthcare | dual-energy spectral CT scanner | |
AW Volumeshare5 | GE Healthcare | dual-energy spectral CT workstation | |
Siemens Inveon micro-PET/CT | Siemens Preclinical Solution | positron emission tomography/ computed tomography scanner |
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Inveon Acquisition Workplace | Siemens Preclinical Solution | PET-CT workstation |