Name: gene regulation and targeted therapy in gastric cancer peritoneal metastasis: radiological findings from dual energy ct and pet/ct
Uploaded: 2018-01-22T14:00:00
Description: Watch this Scientific Journal Video about Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT at JoVE.com

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.

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
<ol>
	<li>Divide a moderately differentiated SGC-7901 human gastric cancer cell line into an SGC-7901/sFRP1 group and an SGC-7901/vector group. Culture the two groups of cells separately in RPMI...

<ol>
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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 ...

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 TGF&#946;, its downstream targets, and TGF&#946;-mediated EMT8. Previous studies have demonstrated that the TGF-&#946;1 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-&#946;1 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&#39; 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-&#946;1 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.

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			<td height="21" style="height:21px;width:247px;">normal saline</td>
			<td style="width:389px;">HUNAN KELUN PHARMACEUTICAL CO,LTD</td>
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			<td height="21" style="height:21px;width:247px;">BALB/c nude mice&#160;</td>
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			<td height="30" style="height:31px;width:247px;">SGC-7901&#160; cells</td>
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			<td height="21" style="height:21px;width:247px;">AW Volumeshare5</td>
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			<td>dual-energy spectral CT workstation</td>
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			<td height="21" style="height:21px;width:247px;">Siemens Inveon micro-PET/CT</td>
			<td style="width:389px;">Siemens Preclinical Solution</td>
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			<td>positron emission tomography/ 
			computed tomography scanner&#160;</td>
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			<td height="21" style="height:21px;width:247px;">Inveon Acquisition Workplace</td>
			<td style="width:389px;">Siemens Preclinical Solution</td>
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			<td>PET-CT workstation</td>
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gene regulation and targeted therapy in gastric cancer peritoneal metastasis: radiological findings from dual energy ct and pet/ct

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-&#946;1 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.

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 me...

Watch this Scientific Journal Video about Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT at JoVE.com

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

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 ...

The overall goal of this non-invasive imaging procedure is to connect research on gastric cancer peritoneal metastasis and the mechanisms of tumorigenesis and to monitor tumor growth and the effects of therapeutic interventions on gastric cancer. This method can help answer some key questions in a tumor research feud, such as how the peritoneal metastasis mechanism functions and how to perform early diagnosis, treatment, response monitoring and prognosis. The main advantage of this technique is that it can identify the region and the metastasis process of gastric cancer and it can monitor the kidneys in response to treatments.To begin this procedure establish the gastric cancer peritoneal metastasis animal model and set up the dual energy CT scan as outlined in the text protocol. After selecting and anesthetizing the experimental mice, click the new patient icon in the DECT program. Input the mouse's basic information including it's patient ID and name.Then in the user protocol section click abdomen protocol and select the animal DECT scan protocol to enter the operation interface. Move each anesthetized mouse onto an animal fixture platform in the supine position. Using tape fix the mouses tail to make sure it does not bend.Sterilize the tail with alcohol. After this move the CT scan bed so that the external positioning line laser is over the mouse's lower abdomen. When the positioning is complete click the reset button.Next click confirm. Follow the flashing order of the buttons on the keyboard to complete the scout scanning. Then select the next series icon and enter the interface for non-enhanced scanning.In the right screen set the start location and end location on the scout views to define the scan range. Maintain the same range in lateral scout and AP scout and cover the entire body volume of the animal. Click the confirm icon and then follow the flashing order of the buttons on the keyboard to complete the scout scanning.After this inject each mouse with iopamidol through the tail vein. Click next series to perform enhanced scanning. Set the start location and end location as before during the non-enhanced scan.Next click the confirm icon and follow the flashing order of the buttons on the keyboard to complete the dynamic enhanced scans, including arterial phase, portal phase and delayed phase. After this click end exam to exit the scanning interface. The image series will automatically be uploaded to the workstation.First locate the mice series on the DECT workstation interface. Select the plus C mono phase series list. Open the GSI volume viewer.And select the GSI VV general protocol from the GSI protocol manager interface. Then click the view type active annotation in the upper left corner of the image view ports and select coronal orientation from the drop down menu. Other orientation images like transverse or sagittal position can be selected from this drop-down menu as well.For one image view port click the volume one active annotation and select mono volumes from the drop-down menu. In a second image view port select iodine water volumes. Click and hold the left mouse button to drag the image from the iodine water view port to the mono view port.Check the mix the views box to get the color fused images. After this click and drag from the center of the image scroll icon to observe the images. Save any that show positive results as color fused images.To begin set up the micro pet CT imaging protocol as outlined in the text protocol. After injecting the FDG and anesthetizing move the animal to the entrance of the pet CT scanner. Click the laser icon from the toolbar viewer and use the touch pad control interface to move the bed so that the mouse's abdomen is located in the center of the pet and CT fields of view.In the laser aligned window select the first scan type as CT scan with pet acquisition included in the workflow as the option. Next open the scout view window and acquire a scout view XR radiograph. Adjust the position of the animal bed so that the CT center field of view is located at the center of the mouse's body.Select the prepared micro pet CT protocol. Input the number of mice to be successively imaged in the pop-up window. Click the set up option and enter the weight.Then click the set up option again and follow the pop up window instructions to complete the set up. Click the start workflow icon to start scanning. After the scanning is complete evaluate the quality of the acquired CT and pet images.Transfer the data through the network to the post imaging analysis. Next open the pet CT workstation software and import the CT and pet image series data. In the registration window click the general analysis option to register CT and pet images together.Then we click the button in the tools application to adjust the window and level of images. Under the review window choose the sky model to show a perfect alignment between the CT and pet images. In the region of interest quantification window identify the peritoneal nodules with references provided by the co-registered images.Draw the region of interest or ROI with tools over the fused images. Edit the size and shape of the ROI and record the maximum standard uptake value. Then output and save the selected merged images.In this study, DECT and pet CT scanning are performed on nude mice after two weeks of cell lined injections. GSI images yielded excellent results for displaying subcutaneous metastasis beyond the contour of the abdomen for the SFRP1 over expression group. And metastasis with peripheral enhancement is confirmed by color scale image.The acquired pet CT images depict focally abnormal FDG uptake of metastasis, including in the peritoneal and subcutaneous metastasis. The gross specimen and histological section further illustrate that peritoneal metastasis and large subcutaneous metastasis. The abdominal cavity in the SFRP1 empty loading group contains no visible lesions, obvious abnormal enhancements or high FDG uptake.However, the gross specimen and histological results confirm that implantation of this group was successful. Non-invasive imaging scans for mice treated with transforming growth factor beta one depict obvious enhancement and focal abnormal fludeoxyglucose uptake of metastasis. The gross specimens for this treatment group are seen to have only eight nodules of peritoneal metastasis with diffused distribution in the abdominal cavity.However, mice in the control group that was given normal saline also showed visible legions and focally abnormal uptake of metastasis. These gross specimens are seen to have 22 nodules of peritoneal metastasis and the local metastasis nodules were adherent to the abdominal cavity. We are attempting this procedure.It is important to make sure that the time period of each experiment or arrangement is reasonable. And performance study has the necessary animal For researchers in a feud of oncology and medicine to research the metastasis mechanism and prognosis evaluation for other tumors like ovarian cancer or rectal cancer. After watching this video you should with understanding of how to use a QCT in a part of CT to access the process of tumor peritoneal metastasis affected by and the treatment efficacy with Don't forget that working with imaging can be extremely hazardous and the precautions such as should always be taken while performing this procedure.

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Research

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JoVE Journal

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Cell Biology

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Unit 1

Cancer

SCIENTISTS IN ACTION

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13.
[18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25.
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography FDG-PET/CT

There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...