이 연구는 DOD 유방암 IDEA 수상에 의해 부분에서 지원됩니다 W81XWH - 08 - 1-0583 및 NIH / NCI CA141348 - 01A1 (DZ)와 FAMRI 임상 과학자 수상 (DS). 이미징 인프라 U24 CA126608와 시몬스 암 센터 (P30 CA142543)와 NIH 1S10RR024757 - 01에 의해 부분적으로 지원 남서 작은 동물 이미징 연구 프로그램에 의해 제공됩니다.

모든 동물의 절차는 텍사스 대학의 남서부 의료 센터의 기관 동물 케어 및 사용위원회에 의해 승인되었습니다. 1. 체외 HIF - 1α bioluminescence 분석에서 <ol><li> 재료 및 방법 : <ul><li> 인간 전이성 유방암 세포주 MDA - MB231은 소설 HIF - 1 - 의존 리포터 유전자로 transfected, 5HRE - ODD 룩은 박사 하라다에 의해 생성되었다. </li><li> hypoxic 조건에서 산소에 의존 열화 도메인 (ODD) 루시페라제 융합 단백질의 향상된 표현은 hypoxia - 응답 요소 5 부 (5HRE)에 의해 구동됩니다. ODD의 존재는 normoxic 조건에서 매우 낮은 배경 루시페라제 활동 결과 ODD 룩 단백질의 저하가 발생합니다. 따라서이 소설의 시스템은 실시간 영상으로 종양의 hypoxic 영역을 감지하는 데 사용할 수 있습니다. 이 5HRE - ODD 룩 표현 벡터의 공사 하라다 외 1,2에 의해보고되었습니다. </li></ul></li><li> normoxia 또는 hypoxia에 따라 문화 세포 : <ul><li> 10% 태아 소 혈청 (FBS) DMEM의 1 % 글루타민, 400 μg / G418의 ML의 항생제, 1 % 페니실린 / 스트렙토 마이신을 포함하는 배지에서 재조합 MDA - MB231 세포를 유지합니다. </li><li> 체외 HIF - 1α bioluminescence 분석, 플레이트 3에 대한 X 10 2 백 6 자 요리의 각 우물에 5HRE - ODD 룩 벡터를 표현 5 MDA - MB231 세포. </li><li> normoxic 조건 (21 % O 2)에 따라 다른 접시를 유지하면서 세포가 하룻밤 배양 후 접시 벽을 첨부하고 hypoxia 연구 hypoxia 챔버 (Billups - 로텐버그 주식 델마, CA) 하나의 요리를 전송할 수 있습니다. </li><li> 가스 실린더와 입구 포트 튜빙을 연결하여 0.1 % O 2 챔버 및 가스 챔버를 재조 립. 참고 : 입구와 출구 포트 클램프 모두가이 절차를 수행하는 동안 열려 있어야합니다. </li><li> 챔버가 플라스틱 클램프를 닫아 정화 및 인감 챔버 후 가스 소스를 분리하십시오. </li><li> 5% CO 2와 37 ° C 배양기에서 챔버를 놓습니다. 참고 : 챔버는 문화의 과도한 증발을 방지하기 위해 humidified해야합니다. 챔버 20 ML 멸균 물 - 이것은 10 배치하여 수행할 수 있습니다. </li></ul></li><li> Bioluminescence 분석 : <ul><li> 24 시간 보육 후, 미디어를 제거하고 얼음 차가운 PBS (2X)로 빠르게 세포를 씻으십시오. </li><li> Luciferin 100 μl (골드 생명 공학, 세인트 루이스, MO)와 차가운 PBS 1 ML을 추가합니다. </li><li> 다양한 노출 시간 (1, 30, 60, 180 s)와 BL 영상 (IVIS 스펙트럼 시스템, 캘리퍼스 생명 과학, Hopkinton, MA) 취득. </li><li> 각각이 잘 살아있는 이미지 소프트웨어 (캘리퍼스 생명 과학)를 사용하여 빛의 강도를 측정합니다. </li></ul></li></ol> 2. 유방암 뇌 전이 모델의 구축 <ol><li> MDA-MB231/5HRE-ODD-luc 세포의 준비 <ul><li> 10% FBS, 1 %의 글루타민, 1 % 페니실린 / 스트렙토 마이신을 포함 DEME 매체 MDA-MB231/5HRE-ODD-luc 세포를 검색과 문화. </li><li> 2~3일 모든 매체를 교체합니다. Tripsinize들이 80 % 합류에 도달하면 세포를 씻으십시오. </li><li> 세포의 적절한 숫자를 카운트 4 μl 볼륨 10 5 세포의 최종 농도 25 %의 Matrigel (BD Biosciences, 산호세, CA)와 혈청 무료 DEME 매체에서 그들을 resuspend. </li><li> intracranial 주사하기 전에 얼음 장소 세포. </li></ul></li><li> 외과 이식 <ul><li> 여성 누드 마우스 (BALB / C 뉴 / 뉴, 국립 암 연구소, 베데스다, MD) 이전 4~6주에서이 연구에 사용됩니다. </li><li> 마취 (유도 챔버에서 O2 / isoflurane 3%; 박스터 인터내셔널, 디어 필드, IL, 미국에서 isoflurane)와 산소 (1 DM 3 / 분) (1 %) isoflurane 수술 3 중에있는 동물을 유지합니다. </li><li> 오른쪽 정수리 피부 betadine 후 이전 절개 70 %의 알코올로 준비되어야합니다. </li><li> 고속 드릴을 사용 버는 코로나 봉합과 saggital 치료 측면 2mm로 두개골, 1mm 앞쪽에의 오른쪽 반구의 1mm 구멍. </li><li> 4 μl 세포 혼합물 (10 5 셀) 10 μl 해밀턴 주사기를 (해밀턴 회사, 리노, NV) 사용하여 그립니다. 바로 바로 꼬리 diencephalon, 맞춤 제작 32G 해밀턴 바늘을 사용하여 경질의 mater 아래 1.5 mm로 세포를 주사. 탈퇴하기 전에 약 30 s에 대한 장소에서 바늘 보관하​​십시오. 32G 좋은 바늘의 사용은 조직 손상을 최소화합니다. </li><li> 뼈 왁스로 버는 구멍을 입력하고 흡수성 봉합과 두피를 닫습니다. </li><li> 70 % 알코올로 절개 지역을 준비합니다. </li><li> buprenorphine 진통제에게 이틀 동안 매 12 시간을 적용합니다. </li></ul></li></ol> 3. 유방암 뇌 전이에 종양 hypoxia 역학 생체내 bioluminescence 이미징에서 <ul><li> 세로 bioluminescence 이미징을 개시 (IVIS 스펙트럼 시스템) 이주 8 주일에 한번 intracranial 주입 반복 후-10 주. </li><li> 한 번에 세 생쥐 (3 % 유도 챔버에서 O 2 / isoflurane)를 마취 </li><li> 이전 4 자세히 설명한대로 각 마우스의 목 지역에서 subcutaneously, D - luciferin (골드 바이오 120, 최저 80 μl의 총 볼륨의 PBS에서 MG / kg)의 솔루션을 관리할 수 있습니다. </li></ul> D - luciferin는 무독성이며 그대로 혈액 뇌 장벽 (BBB)와 세포 세포막을 관통 3,5 수있는 것으로 표시되었습니다. <ul><li> 이미지 챔버에서 3 쥐를 장소와 산소 (1 DM 3 / 분) (1 %) isoflurane 이미징 동안 같이 유지. </li><li> luciferin 주입 후 5 분 동안은 다양한 노출 시간의 배열 (1, 30, 60, 180 s)와 BL 영상을 획득. </li></ul> 우리의 관측 피크 발광 시간이 목에 지역 3,4에서 luciferin의 피하 관리 후 5 분 관한 것을 보여줍니다. <ul><li> 관심 영역에 절대 광자 카운트 (광자 / 초)를 사용하여 생활 이미징 소프트웨어 (캘리퍼스 생명 과학)와 데이터를 분석 (ROI), 수동으로 뇌의 BLI 신호를 대략적으로 그려. </li><li> 광자 카운트의 플롯 시간 코스 곡선 종양 hypoxia 역학을 나타낼 수 있습니다. </li><li> 바로 지난 BLI 후, pimonidazole, hypoxia 마커 나중에 1 시간을 관리 쥐를 희생하고 두뇌를 해부하다. 최적의 절단 온도 (-10 월) -80에서 중간 및 동결 ° C의 냉장고에있는 모든 두뇌를 삽입할 수 있습니다. 이후 histological Cresyl 보라색 얼룩과 루시페라제, hypoxia 마커 pimonidazole에 대한 immunohistochemical 얼룩과 HIF - 1α는 영상 관찰을 검증하기 위해 수행. </li></ul> 4. 대표 결과 : 마찬가지로 Fig.1에 나타난, 상당히 높은 BLI 신호는 24 시간을위한 hypoxic 챔버 (1 % O 2) incubated transfected 세포 후에 관찰되었다. 약한 빛의 방출은 normoxia 아래의 제어 세포에서 관찰되었다. 이것은 HIF - 1α를 overexpress 수있는 세포 스트레스 신호를 유도 3 × 10 5 세포의 24 시간 문화 후 콩나물 세포 인구에서 발생할 수 있습니다. 그럼에도 불구하고, 생체내 BLI 결과 종양 hypoxia와 루시페라제 표현 사이 colocalization을 보여주 immunohistochemical 얼룩에 의해 확인되었다. 노출 시간의 자동 배열은 긴 노출 시간과 약한 신호의 포착을 용이하게 이미지의 시리즈, 그리고 포화 CCD없이 짧은 시간을 사용하여 강한 신호를 지속적으로 인수 가능합니다. <img alt="그림 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> 체외 HIF - 1α bioluminescence 분석에서는 그림 1 끝줄 :. 24 hypoxia 챔버에서 6 - 잘 요리 (0.1 % O 2)의 각 우물에 incubated 3 × 10 5 MDA-MB231/5HRE-ODD-luc 세포 HR은 매체 전에 제거 씻어 1 ML PBS로 대체되었습니다. 100 μl luciferin 각 우물에 추가되었습니다 직후, BLI는 노출 시간의 배열 (1, 30, 60, 180의)로 인수되었다. 강력한 발광은 대표적인 우물에서 관찰되었다 아래 행은 :. 컨트롤로서, 3 × 10 5 세포는 약한 빛을 방출 normoxia (21 % O 2)에 따라 incubated. <img alt="그림 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> 종양 hypoxia 역학의 생체내 bioluminescence 이미징 그림 2. 마우스 두뇌의 오른쪽부터) 약한 빛 신호는 먼저 시각 오주 MDA-MB231/5HRE-ODD-luc 유방암 세포 주입 후 intracranial되었다. 증가 광학 신호가 증가 종양의 hypoxia을 나타내는 추가 6주 동안 관찰했다. B) 줄거리는 빛의 신호의 양적 광자 카운트의 시간 코스 곡선을 보여주었다. <img alt="그림 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> 루시페라제 및 immunohisto - 화학 염색법에 의해 감지 hypoxia의 그림 3 Colocaliztion가. OCT에 포함된 유방암 MDA-MB231/5HRE-ODD-luc의 전이를 베어링 냉동 마우스 뇌가 sectioned되었습니다. 10 μm의 섹션, hypoxic 마커, pimonidazole와 immunostained 방지 루시페라제 염색법에 의해 감지 루시페라제 표현과 spatially 연관시키는 것은 발견 hypoxia의 intratumoral 이질을 공개했다. 스케일 바, 100 μm의.

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관심 없음 충돌 선언하지 않습니다.

유방암 뇌 전이는 단계 IV에서 유방암 환자의 30 %에서 발생합니다. 이것은 높은 병적 상태와 사망률과 관련하여 13개월 6 중앙값 생존을 가지고있다. 그 intracranial 개시 및 진행뿐만 아니라 pathophysiological 프로필에 대한 우리의 이해를 촉진하기 위해이 임상적으로 치명적인 질병을 모방하기 위해 적절한 동물 모델을 필요가있다. 여기, 우리는 직접 마우스 두뇌로 인간 유방암 세포를 주사하?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> D - luciferin </td><td> 골드 생명 공학 </td><td> L - 123 </td><td> 120 생체내 연구에 80 μl의 총 볼륨의 PBS에서 MG / kg </td></tr><tr><td> Isoflurane </td><td> 벡스터 인터내셔널 </td><td> 1001936060 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td></td></tr><tr><td> 해밀턴 주사기 </td><td> 해밀턴 회사 </td><td> 1701 </td><td></td></tr><tr><td> 32G 해밀턴 바늘 </td><td> 해밀턴 회사 </td><td> 7803-04 </td><td></td></tr><tr><td> Hypoxia 챔버 </td><td> Billups - 로텐버그 주식 회사 </td><td> MIC - 101 </td><td></td></tr><tr><td> Bioluminescence 이미징 시스템 </td><td> 캘리퍼스 생명 과학 </td><td> IVIS 스펙트럼 시스템 </td><td></td></tr><tr><td> G418 </td><td> 피셔 과학 </td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

이곳은 종양 hypoxia은 악성 진행을 촉진하고 부정적인 치료 반응에 영향을 미치는에 중요한 역할을 것을 인식됩니다. 때문에 이러한 뿌리 깊은 orthotopic 종양에서 모니터링하는 효율적인 수단의 부족 악성 뇌 종양의 intracranial 개발하는 동안 생체내, 종양 hypoxia의 현장에 대한 약간의 지식이있다. 루시페라제 유전자를 표현하는 살아있는 세포에 의해 방출되는 빛의 감지에 따라 Bioluminescence 이미징 (BLI)는, 빠르게 잠복기 동물 연구에서 치료에 대한 응답으로 종양의 성장이나 종양 크기 변화를 평가하기 위해, 특히 암 연구에 채택되었습니다. 또한, 발기인 순서의 제어하에 기자의 유전자를 표현하여, 특정 유전자 발현이 BLI에 의해 비 invasively 모니터링할 수 있습니다. hypoxic 스트레스에서 신호 응답은 여러 유전자의 전사를 드라이브에 주로 hypoxia inducible 인자 - 1α (HIF - 1α)를 통해 중재 수 있습니다. 따라서, 우리는 안정적으로 인간 유방암 세포 MDA - MB231 (MDA-MB231/5HRE-ODD-luc). 체외 HIF - 1α bioluminescence 분석에 의해 수행에 transfected HIF - 1α 기자 구조, 5HRE - ODD 룩을 사용한 normoxia에있는 세포 (21 % O 2) 제어 역할을하면서, BLI 전에 24 시간을위한 hypoxic 챔버 (0.1 % O 2)에 transfected 세포를 잠복기. hypoxia에서 세포에 대한 관찰 상당히 높은 광자 플럭스은 normoxia 이들에 비해 자사의 발기인으로 증가 HIF - 1α 바인딩 (HRE 요소)을 제안합니다. 셀은 유방암 뇌 전이 모델을 수립하기 위해 마우스 두뇌에 직접 주입됩니다. 종양 hypoxia 역학의 생체내 bioluminescence 이미징에서 주입 후 2 wks을 시작하고 일주일에 한 번 반복됩니다. BLI는 증가 intracranial 종양의 hypoxia을 나타내는, 종양 진행으로 두뇌에서 빛을 신호를 증가 보여줍니다. Histological과 immunohistochemical 연구는 생체내 이미징 결과를 확인하는 데 사용됩니다. 여기, 우리는 체외 HIF - 1α bioluminescence 분석, intracranial 종양의 hypoxia를 모니터링하는 생체내 bioluminescence 이미징에의 누드 마우스와 응용 프로그램에서 유방암 뇌 전이의 수술 설립의 방법을 소개합니다.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

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

hypoxia inducible 인자 - 1α 활동 Bioluminescence 이미징은 유방암 뇌 전이의 마우스 모델에서 종양 intracranial hypoxia 개발을 모니터에 적용됩니다.

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 ...

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JoVE Journal

Medicine

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

19.5K 조회수.  University of Texas Southwestern Medical Center. hypoxia inducible 인자 - 1α 활동 Bioluminescence 이미징은 유방암 뇌 전이의 마우스 모델에서 종양 intracranial hypoxia 개발을 모니터에 적용됩니다.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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의학

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Orthotopic 마우스 뇌종양 모델의 조합 방사선 치료

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotactic Intracranial 주입 및<em&gt; 생체에</emGlioblastoma의 Multiforme의 마우스 모델 시스템에서 종양 Xenografts의&gt; 발광 생물 이미징

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

유방암의 마우스 모델에서 종양 Microenvironment에 약물 응답의 영상을 라​​이브

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

종 MRI에 이어 유방암 뇌 전이 마우스 모델을 개발하기 위해 초음파 영상 유도 심장 내 주입

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

유방암의 소설 형질 전환 마우스 모델 : 아데노 바이러스 크레와 유관 상피의 대상으로 전이성 유방암의 개시

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

형광 SAPC-DOPS Nanovesicles을 사용하여 뇌 종양 및 관절염의 생체 광학 이미징

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...