Name: monitoring tumor metastases and osteolytic lesions with bioluminescence and micro ct imaging
Uploaded: 2011-04-14T13:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging at JoVE.com

세로 이미지는 질병의 진행을 따라하거나 치료의 효과를 측정하기 위해 사전 임상 연구에 사용됩니다. 종양학에서는 광학 방법은 종양 성장을 모니터하고 이러한 연구의 각 시점에서 세포 성장이나 유전자 발현의 정확한 quantitation를 제공하기 위해 엄격한 도구를 제공합니다. 해부 학적 변경 microCT 같은 고해상도 기법을 사용하여 측정할 수 있지만, 세로 이미지에 대한 낮은 X - 선 선량은 생물 학적 유물을 피하기 위해 사용해야합니다. 광학과 microCT 이미지는 최대 정보가 동물 모델에서 추출한 것을 보장 기능과 해부 학적 데이터의 조합을 제공하기 위해 공동으로 등록할 수 있습니다. 1. 셀 준비 <ol><li> 캘리퍼스는 마우스 모델에서 미리 임상 연구를위한 루시페라제 표현 암 세포 라인의 범위를 제공합니다. </li><li> MDA - MB - 231 - 륙 - D2H2LN은 생체내에 tumorgenesis의 광학 지표로 사용할 수있는 루시페라제 유전자를 표현하는 인간 내유 종양 세포 라인입니다. 이 세포 라인은 D3H1 내유 지방 패드 종양에서 유래 자연 림프절의 전이에서 만들어지고 적극적으로 metastases를 형성하는 것으로 알려져 있습니다. </li><li> 세포는 쉽게 선택 마커에 대한 필요가 표준 미디어 성장 병원균이없는 냉동 문화로 제공됩니다. </li><li> 주입하기 전에 동물로 루시페라제 활동을 확인하려면 90% 합류 플라스크는 trypsinization의 수확이다. 루시페라제 활동 microtiter 접시에 50,000 세포를 분배 및 시리얼 희석를 수행하여 측정됩니다. </li></ol> 2. 동물 세포의 Intracardiac 분사 <ol><li> 주입하기 전에 동물 3 % isoflurane을 사용하여 anesthetized 있습니다. </li><li> 1-3000000 세포가 좌심실에 50ul 볼륨에 주입하고 있습니다. 세포 현탁액은 150ug/ml D - Luciferin가 분사 기술을 확인하는가 포함되어 있습니다. </li><li> 올바른 intracardiac 사출 마우스를 확인하려면은 IVIS 시스템에서 몇 군데 있습니다. 체내 BLI 신호를 보여 생쥐가 제대로 주입되었습니다. 신호화된 경우 세포는 중심에 정확하게 주입되지 않았습니다. </li></ol> 3. BLI 영상은 metastases을 모니터 <ol><li> 영상 셔틀 IVIS 스펙트럼과 QuantumFX microCT 시스템 모두에서 데이터 캡처를위한 마우스를 지원합니다. </li><li> 셔틀 이미징에 대해 같은 위치에 동물을 유지하는 데 도움이 공동 등록 자동에 투입 fiducial 신호를 포함합니다. 챔버는 마취를 유지하기 위해 isofluorance의 기화기에 연결됩니다. </li><li> 생활 이미지 소프트웨어 4.1 이미징 마법사가 자동으로 노출 시간, F - 중지하고 비닝을 선택하고 양적 데이터 수집을 보장하는 데 사용할 수 있습니다. </li><li> 여러 이미지를 획득하여 실험의 특성에 따라 초 개월에 걸쳐 종단 연구에 비교할 수 있습니다. </li><li> 세포의 6 일 포스트 주사, IVIS 스펙트럼과 BLI 영상 아홉 생쥐에서 7 뼛속까지 metastases을 보여줍니다. </li></ol> 4. 마이크로 CT를 사용 osteolytic 병변의 길이 방향 모니터링 <ol><li> BLI를 사용하여 보이는 맑은 골 metastases을 보여주 생쥐는 양자 FX 마이크로 중부 표준시 시스템에서 몇 군데 있었다. </li><li> 퀀텀 FX 마이크로 중부 표준시 시스템은 잠복기 연구에서 진정한 길이 마이크로 CT를 할 수있을만큼 충분 낮은 X - 선 선량에서 높은 품질의 이미지를 제공합니다. </li><li> 두 다리 뼈 저하는 길이 방향 분안에이 시스템을 사용하여 모니터링할 수 있습니다. </li><li> 마우스는 isoflurane 3퍼센트로 anesthetized되고 IVIS 스펙트럼 시스템에서 직접 전송하실 수 있습니다. </li><li> 두 60mm 이미지는 전체 동물보기를 캡처 함께 맞춘 있습니다. </li><li> 30mm FOV는 영상 고해상도의 무릎 관절에 이상적입니다. </li><li> 뼈 metastases를 측정하고 수치만큼 충분히 높은 해상도를 가진 하나의 검사는 막 18 초 소요 단 13mGy의 복용을 제공합니다. </li><li> 생쥐는 동물의 종양 성장이나 건강에 영향을 미치지 않고 마이크로 중부 표준시 시스템 36일 8 시간까지 몇 군데 있었다. </li><li> 마이크로 중부 표준시 이미지는 뼈 leisions가 10 일째에 볼 수 있었고, BLI 신호의 증가에 대응하는, 매우 빠르게 진행되고 것으로 나타났다. </li></ol> 5. BLI와 uCT의 3D는 공동 등록 및 분석 <ol><li> 일단 이미지는 3 차원 재건에 공동 등록 수는 IVIS 스펙트럼 및 양자 FX 마이크로 중부 표준시 시스템 데이터 모두에 생포되었습니다. </li><li> MicroCT 이미지는 DICOM 파일로 내보낼와 생활 이미지 4.1 3D 멀티 양상 도구에로드됩니다. </li><li> 생활 이미지 소프트웨어의 광 신호를 재구성 치수 셋을 DLIT 프로그램을 실행합니다. </li><li> 소프트웨어의 fiducial 도구는 마이크로 중부 표준시 이미지로 광학 BLI 신호를 공동 등록하는 데 사용됩니다. </li><li> Coregistered 데이터 세트는 여기에 우리가 뼈에 osteolytic 병변의 발전과 함께 전이성 종양의 성장을 추적하는 질병 진행하는 동안 여러 개의 매개 변수를 모니터링하는 양적 분석하실 수 있습니다. </li> &lt;/ 올&gt;

<ol>
	<li>Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J., Purchio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Human+Breast+Cancer+Cell+Lines+that+Permit+Rapid+and+Sensitive+in+vivo+Detection+of+Mammary+Tumors+and+Multiple+Metastases+in+Immune+Deficient+Mice.">Bioluminescent Human Breast Cancer Cell Lines that Permit Rapid and Sensitive in vivo Detection of Mammary Tumors and Multiple Metastases in Immune Deficient Mice.</a> Breast Cancer Research. 7, R444-R454 (2005).</li><li>Cowey, S., Szafran, A. A., Kappes, J., Zinn, K. R., Siegal, G. P., Desmond, R. A., Kim, H., Evans, L., Hardy, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Metastasis+to+Bone:+Evaluation+of+Bioluminescent+Imaging+and+MicroSPECT/CT+for+Detecting+Bone+Metastasis+in+Immunodeficient+Mice.">Breast Cancer Metastasis to Bone: Evaluation of Bioluminescent Imaging and MicroSPECT/CT for Detecting Bone Metastasis in Immunodeficient Mice.</a> Clinical and Experimental Metastasis. 24, 389-401 (2007).</li><li>Labrinidis, A., Hay, S., Liapis, V., Ponomarev, V., Findlay, D. M., Evdokiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronic+Acid+Inhibits+Both+the+Osteolytic+and+Osteoblastic+Components+of+Osteosarcoma+Lesions+in+a+Mouse+Model.">Zoledronic Acid Inhibits Both the Osteolytic and Osteoblastic Components of Osteosarcoma Lesions in a Mouse Model.</a> Clinical Cancer Research. 15, (2009).</li><li>Kaijzel, E. L., Pluijm, G. v. a. n. d. e. r., Lowik, C. W. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Body+Optical+Imaging+in+Animal+Models+to+Assess+Cancer+Development+and+Progression.">Whole-Body Optical Imaging in Animal Models to Assess Cancer Development and Progression.</a> Clinical Cancer Research. 13, 3490-3497 (2007).</li><li>Minn, A. J., Kang, Y., Serganova, I., Gupta, G. P., Giri, D. D., Doubrovin, M., Ponomarev, V., Gerald, W. L., Blasberg, R., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Organ-Specific+Metastatic+Potential+of+Individual+Breast+Cancer+Cells+and+Primary+Tumors.">Distinct Organ-Specific Metastatic Potential of Individual Breast Cancer Cells and Primary Tumors.</a> The Journal of Clinical Investigation. 115, (2005).</li><li>Xu, H., Christensen, A., Kuo, C., Nilson, D., Lim, E., Whalen, J., Kim, J. B., Zhang, N., Singh, R., Oldfield, S., Troy, T., Rice, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-registration+of+in+vivo+Optical+Tomography+and+Micro-CT+for+Longitudinal+Studies.">Co-registration of in vivo Optical Tomography and Micro-CT for Longitudinal Studies.</a> Word Molecular Imaging Congress. , (2010).</li></ol>

저자는이 문서에 등장하는 악기를 만드는 칼리퍼 생명 과학의 직원입니다.

microCT과 작은 동물 이미징은 같은 종양 개발 (..; Labrinidis 외, 2009 Cowey 외 2007)과 같은 병적인 조건 아래에서 모니터링 osteolytic 병변에 적용되었습니다. 루시페라제 분류 종양의 bioluminescence 이미징 널리 사전 임상 채택되었습니다로서, bioluminescence과 microCT 이미지의 공동 등록 bioluminescence 신호 (Kaijzel 외. 2007)의 해부 학적 위치의 더 나은 정의를 제공했습니다. 루시페라제 표시 유방 종양 세포 라인 MDA - MB - 231 - 륙 - D3H2LN와 함께 이전 연구는 내부 심장 분사 다음과 같은 뼈와 osteolytic 병변의 발전에 metastases 보여 (젠킨스 외 2005;. 민 외, 2005). 본 연구에서는, 우리는 체계적 MDA - MB - 231 - 륙 - D3H2LN의 전이 모델과 조합 광학과 낮은 방사선 선량 - microCT 이미징과 함께 종양 metastases과 osteolytic 활동의 종단 연구를 탐험. 우리는 IVIS 스펙트럼 및 양자 FX microCT 시스템 및 달성 bioluminescence 및 중부 표준시 우리가 새롭게 개발한 생활 이미지 4.1 소프트웨어 (헹 외., 2010)와 공동으로 등록 순차 이미징을 수행. IVIS과 Bioluminescence 이미징은 종양이 초기 단계에서 감지 수 있도록 높은 민감도와 종양 세포의 비침습 감지 기능을 제공합니다. 퀀텀 FX와 영상은 뼈 병변 및 자세한 해부 학적 정보의 정확한 3 차원 재구성 수 있습니다. 퀀텀 FX 시스템은 질병 진행의 길이 모니터링을 가능하게 낮은 방사선 선량 이미징을 제공합니다. 뼈 병변 및 종양 metastases의 상관 관계는 공동 등록 이미지에 묘사된되었고, 병변의 개발 multimodality 추적의 이점은 길이 방향 명확하게 증명되었다.

monitoring tumor metastases and osteolytic lesions with bioluminescence and micro ct imaging

뉴 / 뉴 마우스로 MDA - MB - 231 - 륙 - D3H2LN 세포의 다음 intracardiac 전달, 주입 동물에서 개발된 체계 metastases. IVIS 스펙트럼을 사용하여 Bioluminescence 이미징은 종양 신호와 위치를 측정하는 DLIT 재건축 포함하여 전달 절차에 따라 종양 세포의 분포와 개발을 모니터 고용했다. 뼈 조직에 전이성 병변의 개발은 경골에 osteolytic 활동과 병변을 유발하고 대퇴골은 마이크로 CT를 사용하여 길이 방향 평가했다. 이미징은 빠른 이미징과 낮은 X - 선 선량과 양자 FX 마이크로 중부 표준시 시스템을 사용하여 수행되었습니다. 낮은 방사선 선량은 여러 영상 세션까지 LD50 아래 누적 X - 선 조사량을 수행할 수 있습니다. 마우스 이미징 셔틀 장치는 순차적으로 이미지를 IVIS 스펙트럼과 양자 FX 모두 쥐가 bioluminescence 및 중부 표준시 이미지를 모두 정확 동물 포지셔닝을 달성 사용되었다. 광학 및 중부 표준시 데이터 세트는 생활 이미지 4.1 소프트웨어를 사용하여 3 - dimentions 공동 등록되었다. 이 다중 모드 방식은 osteolytic 활동을 동시에 종양 성장과 발전의 가까이 모니터링을 허용합니다.

Watch this Scientific Journal Video about Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging at JoVE.com

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

뼈 전이의 실험 마우스 모델은 루시페라제 내유 종양 세포를 표현 intracardiac 배달 아래 설립되었습니다. 종양 개발 및 osteolytic 병변을 발생은 bioluminescence과 마이크로 중부 표준시 이미징과 길이 방향 감시했다.

Bioluminescence 및 마이크로 중부 표준시 이미징과 함께 종양의 Metastases 및 Osteolytic 조직 손상을 모니터링

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

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Universidad San Sebastian

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비디오: Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

유방암 뇌 전이의 종양 Hypoxia 역학 생체내의 Bioluminescence 이미징에서는 마우스 모델에서

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

연구

의학

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

생체 듀얼 기판 발광 생물 이미징에

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

다 발성 골 수 Xenografts 노 그 쥐에서의 복합 발광 및 양자 방출 단층 촬영/컴퓨터 단층 촬영 영상

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

암 연구학

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.

자기 공명 영상, 체적 계산의 Tomography 및 초음파를 이용한 유방암의 뼈 전이의 누드 쥐 모델에서 Angiogenesis의 멀티 모달 이미징

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

뼈 전이의 모델

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

꼬리 정맥 전이 검사의 결합된 사용 및 생체 내 실시간 생체 내 이미징을 결합하여 폐암 전이성 식민지화 및 폐의 부담을 정량화

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

원발성 뼈 종양 및 뼈에 이식된 고형 종양을 통한 뼈 전이 모델링

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

생체 발광을 사용하여 마우스의 유방암 성장 및 전이성 콜로니 형성 모니터링

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>orthotopically 주입된 유방 종양 세포의 자발적 폐 전이를 측정하기 위한 In vivo imaging

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.