우리는 박사 부부에게 감사의 말씀을 전합니다. 우리 MDA-MB231Br 세포를 제공하는 다이앤 팔미 에리와 NCI의 패트리샤 스티. 우리는 기술 및 평등 한 지원을 위해 박사 랄프 메이슨 씨 제이슨 Reneau과 양 라몬 로페스 감사합니다. 이 작품은 DOD IDEA 어워드 W81XWH - 08 - 1-0583와 W81XWH-12-1-0317에 의해 부분적으로 지원되었다. MRI 실험은 고급 이미징 연구 센터, NIH BTRP # 1 P41-RR02584 시설에서 수행하고, 초음파 유도 심장 내 주사 1S10RR02564801에서 VisualSonics 베보 (770)를 수행 하였다.

모든 동물의 절차는 텍사스 대학 남서 의료 센터의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. MDA-MB231/Br-GFP 세포의 준비 <ol><li> 10 % FBS, 1 % 글루타민 및 1 % 페니실린 / 스트렙토 마이신을 함유하는 DMEM 배지 (친절의 Drs. 팔미 및 스티, NCI에 의해 제공) MDA-MB231/Br-GFP 셀을 검색하여 배양. </li><li> 세포와 매체의 색상의 상태를 관찰하고, 2-3 일마다 중간 교체합니다. </li><li> 를 Trypsinize 및 단계에 설명 된대로 80 %의 합류가 1.3.1-1.3.5에 도달하면 세포를 수집 <ol><li> 완전히 오래된 매체를 제거하고 부드럽게 세포를 씻어 5 ㎖의 PBS (1X)를 추가합니다. 접시에서 PBS를 제거합니다. </li><li> 접시에 1.5 ㎖의 트립신을 추가, 모든 세포는 트립신에 의해 보호됩니다 확인하기 위해 부드럽게 요리를 기울이십시오. 다시 세포 배양기에 접시를 넣고 1 분 동안 37 ° C에서 보관. </li><li> 인큐베이터 밖으로 접시를 타고 관찰세포를 확인하기 위해 광학 현미경으로 세포를 접시에서 분리. </li><li> 트립신의 효력을 정지 3 ㎖ 매체를 추가합니다. 원심 관에서 세포 혼합액을 모은다. 5 분 동안 2,000 rpm에서 혼합물을 원심 분리기. </li><li> 신중하게 매체를 제거하고 균일 할 때까지 5 ㎖ 혈청 배지에서 세포를 재현 탁. </li></ol></li><li> 세포의 적절한 수를 계산하고 100 μL 부피의 1.75 × 105 세포의 최종 농도로 무 혈청 DMEM 배지에이를 재현 탁. <ol><li> 50 ㎕의 세포 혼합물을 50 ㎕의 트리 판 블루를 추가합니다. 혼합 한 후, 10 ㎕의 혼합물을 조심스럽게 세포 계수 슬라이드에 추가 할 수 있습니다. 정확한 세포 수의 추정을 보장하기 위해 여러 개의 샘플을 사용합니다. </li><li> 셀 카운팅 슬라이드, 한번에 하나의 단부와 셀 카운터 자동 카운트 시작의 막대기. 모든 계산 결과의 평균을 가지고 총 세포 수를 계산합니다. </li><li> 다시 세포를 원심 분리기와 그들을 재현 탁볼륨 μL 1.75 × 105 cells/100의 최종 농도 초래할 무 혈청 배지의 적절한 볼륨. </li></ol></li><li> 이전에 심장 내 주입에 얼음 장소 세포. </li></ol> 2. 초음파 이미징 안내 심장 내 주입 <ol><li> 이전 6-8주 사이 여성 누드 마우스 (BALB / C 뉴 / 뉴)를 사용합니다. </li><li> 영상 시스템에 로그인합니다. 트랜스 듀서 (704) (40 메가 헤르츠)를 초기화합니다. </li><li> 새로운 연구를 시작하고 정보를 입력합니다. </li><li> 마취 (유도 챔버에서 3 % isoflurane/100 %의 O 2)와 코 콘을 통해 모든 절차를 수행하는 동안 100 % O 2 (1 DM 3 / 분)에 이소 플루 란 (2 %)와 동물을 유지한다. 금단 반사가 발가락 핀치 관찰되지 않은 경우 마취가 확인됩니다. </li><li> 37 ° C로 영상 테이블의 온도를 설정 앙와위에서 가열 된 영상 테이블에 마취 된 마우스를 테이프. </li><li> 메신저에 앞서 37 ° C에서 초음파 젤을 유지노화. 마우스의 가슴에 젤을 적용합니다. </li><li> 홀더에 트랜스 듀서를 장착합니다. 원하는 영상의 깊이에 도달 할 때까지 트랜스 듀서를 낮 춥니 다. 좌심실이 랜드 마크로서 상행 대동맥 (그림 1A)로 확인 될 때까지 단계를 이동합니다. 좌심실의 밝은 전망이 가시화되는 단계를 잠급니다. </li><li> 22 G 바늘로 1 ML의 주사기로 세포 혼합물의 100 μl를 그립니다. 놓고 마운트 주사기 주사기를 고정합니다. </li><li> 마우스의 가슴을 향해 앞으로 주사기를 이동하고 바늘 끝이 (마우스를 입력하기 전에)보기의 이미지 필드가 될 때까지 조심스럽게 좌우로 그것을 옆으로 이동합니다. </li><li> 좌심실을 목표로 바늘의 높이와 각도를 조절합니다. </li><li> 초음파 영상의지도하에 좌심실로 즉시 피부와 근육 층을 통해 인터 공간에 주사기 바늘을 침투. 왼쪽 ventri에 바늘을 성공적으로 삽입 표시CLE는 주사기에 신선한 동맥혈 (진한 빨강 정맥혈 달리 핑크 색상)의 환류입니다 </li><li> 세포 혼합물을 천천히 (그림 1B)를 주입한다. </li><li> 완료되면, 바늘을 철회 트랜스 듀서를 들어, 적신 거즈와 초음파 젤을 청소하고 테이프를 제거합니다. </li><li> 예열 패드 깨끗한 케이지를 준비합니다. </li><li> 패드에 동물을 배치하고 전체 복구까지 동물을 관찰합니다. </li><li> 동물의 행동 이틀 동안 매 24 시간을 모니터링합니다. </li><li> 일반 조건 및 실험 생쥐의 합병증의 신경 학적 징후는 정기적으로 모니터링된다. </li></ol> 3. 두개 내 종양 개발의 MRI 모니터링 <ol><li> 뇌 전이의 두개 내 개발을 모니터링하는 9.4 테슬라 자석을 사용합니다. </li><li> 이주 종양 이식 후 MRI를 시작하고 최대 세 주 동안 일주일에 한 번 반복합니다. </li><li> 3 % 이소 플루 란과 동물을 진정 유전자에서 그들을 유지RAL 마취 (1.5 % 이소 플루 란). </li><li> 모니터링하고 실험 내내 동물 체온과 호흡 수를 일정하게 유지한다. </li><li> 고해상도 멀티 나선형 (1 mm의 14 슬라이스 두께, 간격) T 1 -과 T 2 강조 관상 후두에 전두엽의 영역을 포함하는 이미지, 다음과 같은 매개 변수를 취득 : T 1 강조 영상을 : 에코 여러 조각 (SEMS)를 스핀, TR / TE = 400 msec/20 밀리 초, 매트릭스 : X 256 256, 평면 해상도 FOV 20 × 20 mm의 : 78 X 78 μm의 2. T 2 강조 영상 : 256 X 256, FOV 20 × 20 mm의 평면 해상도 : 78 X 78 μm의 빠른 스핀 여러 조각 (FSEMS) 시퀀스, TR / TE = 2500 msec/48 밀리 초, 8 에코 열차, 매트릭스를 에코 28.9. 종양 병변은 T 2 강조 영상에서 정상 뇌 조직보다 밝게 나타납니다. </li><li> 종양의 크기는 수동으로 enhan을 요약하여 T 2 강조 영상에서 결정된다우리 에이트 작성한 MATLAB 프로그램을 이용하여 각 이미지의 질량 부를의 cing. 직경이 슬라이스 두께 (1mm)보다 작은 종양의 대부분을 감안할 때, 종양의 크기는 오히려 부피보다 평면 영역으로 표시된다. </li></ol> 4. H &amp; E 염색은 전이 확인 <ol><li> 종양 베어링 뇌를 해부, 바로 다음 MR 스캔 후 쥐를 희생의 OCT 매체에 포함와 C -80 ° 동결 </li><li> 제 10 ㎛ 두께의 관상 뇌의 일련의 저온 유지 장치와 표본. </li><li> 뇌 부분에 H &amp; E 염색을 수행합니다. </li></ol>

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관심 없음 충돌 선언하지 않습니다.

본 연구에서는이 연구 개발 뇌 전이의 모든 동물과없는 동물의 죽음이 관찰되었다 있도록 초음파 영상 유도 좌심실 분사의 정확성을 보장 것을 증명하고있다. 영상 유도 주사의 아름다움은 피부 그리고 마지막으로 좌심실에 바늘 침투의 경로를 모니터링하고 따라 엄격한 해부학 적 랜드 마크를 필요로하는 수동 절차 구별되는 이미지에 따라 조절 될 수 있다는 것입니다. 뇌...

뇌 전이는 성인에서 가장 흔한 두개 내 악성 종양이다. 예후도 적극적인 치료 4-6 개월의 평균 생존에 매우 좋지 않습니다. 유방암 뇌 전이 1-3의 높은 사망률을 가진 세 가지 주요 암 중 하나입니다. 여러 뇌 열대 유방암 라인은 심장 내 또는 intracarotid 주입 4 후 뇌 전이를 개발 할 수있다. 좌심실로 종양 세포의 직접적인 주입은 폐 모세 혈관 베드를 우회함으로써 내장 전이를 최소화하면서 두뇌 전이 형성의 발생을 증가시킬 수있다. MDA-MB231Br 라인은 설치류 모델 5,6에서 뇌 전이를 개발하기 위해 가장 널리 사용되는 인간 유방암 라인 중 하나이다. 다른 많은 연구 4,7와 마찬가지로, 우리는 우리의 이전 연구에서 심장 내 주입의 수동 절차를 수행했습니다. 그러나, 50 %의 성공 속도는 매뉴얼을 얻었다이전의 실험이 실패한 경우 주사와 마우스의 일부가 반복으로서 절차에서 사망했다. 여기서, 우리는 무 흉선 마우스의 좌심실로 뇌 추구 유방암 세포의 주입을 확보 촬상 유도 절차의 사용을 도입하고있다. 종 고해상도 MRI는 뇌 전이의 두개 내 개발을 따라 적용됩니다.

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			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
			<td style="width:87px;"> </td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
		</tr>
	</tbody>
</table>

ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

유방암 뇌 전이가 단계 IV에서 유방암 환자의 30 %에서 발생하는 높은 사망률과 연관된다. 중앙 생존 기간은 단지 6 개월입니다. 그것은 임상 시나리오에서 전이성 세포의 혈역학 적 확산을 모방하기 위해 적절한 동물 모델을 가지고하는 것이 중요합니다. 여기서, 우리는 흉선 누드 마우스의 좌심실로 뇌 열 유방암 세포의 정확한 주입을 안내하는 작은 동물 초음파 영상의 사용을 도입하고있다. 세로 MRI는 두개 개시 및 뇌 전이의 성장을 평가하는 데 사용됩니다. 초음파 유도 심장 내 주입은 정확한 주입 및 이에 높은 성공률뿐만 아니라 보장뿐만 아니라 우리의 이전의 수동 절차에 비해 크게 사망률 감소했다. 생체 고해상도 MRI는 한 작게, hyperintense 다발성 병변의 시각화 수 310 μm의 T에 직경 삼주 후 분사에 이미지를 2 - 가중. FOLLOW 업 MRI는 두개 내 종양의 성장과 뇌 전체에 걸쳐 배포 전이의 증가 수를 보여준다.

MRI (평면 해상도에 78 μm의)의 높은 공간 해상도로, hyperintense 병변 (그림 2) 직경 310 ㎛의 한 작은 식별 할 수 있습니다. 이 연구에서 전이가 매우 작은 괴사와 부종의 개발 때문에 T 2 강조 영상에서 hyperintense 병변이 진정으로 종양의 질량을 나타내는 최소이다. 종관 MRI 연구는 종양 성장의 비 침습적 생체 평가에 허용한다. 도 3에 도시 ?...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

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

유방암 뇌 전이의 마우스 모델은 MDA-MB231/Br-GFP 세포의 초음파 영상 유도 심장 내 주입으로 설정됩니다. 다 초점 두개 내 전이의 개발은 고해상도 9.4 T MRI를 사용하여 세로 방향으로 모니터링하고있다.

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 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K 조회수.  University of Texas Southwestern Medical Center.  유방암 뇌 전이의 마우스 모델은 MDA-MB231/Br-GFP 세포의 초음파 영상 유도 심장 내 주입으로 설정됩니다. 다 초점 두개 내 전이의 개발은 고해상도 9.4 T MRI를 사용하여 세로 방향으로 모니터링하고있다. 

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

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

쥐 모델에서 Transcranial 집중 초음파를 사용하여 혈액 - 뇌 장벽의 MRI를 유도 장애

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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 ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

세로 두 광자 이미징 다음 마우스의 급성 뇌 외상

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

간 전이의 전임상 쥐과 모델

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

마우스의 유방 지방 패드에 유방암 세포의 소성 주입은 종양의 성장을 연구한다.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.