We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.

모든 마우스 실험은 의학의 LSU 학교의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인 된 프로토콜에 따라 수행되었다. 모든 악기는 해부하기 전에 멸균해야합니다. 마우스 유방 종양에서 일차 전지 1. 준비 <ol><li> 유전자 이전 6 설명한 바와 같이 자발적 종양을 개발하는 마우스 유방 종양 바이러스 폴리오 중동 T-항원 (MMTV-PyMT 유전자 변형 마우스)로 설계 한 마우스 모델을 사용합니다. </li><li> 아이소 플루 란 (99.9 %)와 쥐를 희생하고 무균 환경 (그림 1A)을 보장하기 위해 75 %의 에탄올을 스프레이. </li><li> 가위로 목 요도 구멍의 영역으로부터 피부를 엽니 다. 기관 (도 1b)에 쉽게 접근 할 수 있도록 피부 아래에 핀. </li><li> 유방 유방 종양을 분리하고 차가운 멸균 인산 완충 식염수 (PBS) (PH 7.4) (그림 1C)로 씻는다. </li><li> 에 종양 이동멸균 10cm 조직 배양 판은 약 1mm 3 개에 멸균 면도날과 가위로 잘라합니다. 후속 단계는 무균 층류 후드에서 수행되어야한다. </li><li> 37 ° C에서 2 시간 동안 로커에서 1 ㎎ / ㎖의 최종 농도에서 무 혈청 DMEM 배지에 용해 된 콜라게나 다진 종양 조직을 인큐베이션. g X 200에서 3 분 동안 종양 조직 현탁액을 원심 분리기. 참고 : 콜라게나 배양은 조직의 효소 분해에 필수적이다. </li><li> 뜨는을 취소하고 200 x g에서 3 분 동안 원심 분리하여 PBS (PH 7.4)와 침전물을 3 회 반복한다. 최종 세척 후, 상층 액을 가만히 따르다. </li><li> 하면서 하향 경사 37 ° C에서 5 분 동안 트립신 EDTA 2 ㎖와 조직을 재현 탁. 200 XG에 3 분 동안 현탁액을 원심 분리기 및 세척을 반복합니다. </li><li> 10cm 접시에 정상적인 성장 배지 (DMEM, 10 % FBS, 배 펜 / 연쇄상 구균)에서 조직 펠렛을 재현 탁. 변화미디어 다음 3 일 동안 매일. 목적하는 세포 수까지 배양 세포가 얻어진다. 참고 : 약 1 × 7 세포는이 절차에서 격리됩니다. MMTV-PyMT 비교할 때 이러한 MMTV-노이 같은 다른 마우스 유방 종양 모델은, 세포의 낮은 수율을 제공한다. 낮은 수율을 생성 모델을 사용하면, 더 이상 큰 종양 세포 배양으로 시작하는 것이 유리하다. </li></ol> 2. 마우스 폐 추출 폐 전이를 시각화 <ol><li> 폐 추출에 앞서, 간 및 진동판을 제거한다. 폐를 방해하지 않고 갈비뼈을 제거. </li><li> 위의 미세 쌍, 경정맥에 가까운 기관의 상부를 연다. </li><li> 카테터를 사용하여, 폐 (도 2A)를 채우기 위해 기관을 통해 Z-수정 또는 인도 잉크 등의 1 ml의 에이전트를 주입. 포함 파라핀에 폐를 Z-해결. </li><li> Fekete의 솔루션에 인도 잉크를 주입 폐를 드 얼룩(100 mL의 70 % 알코올, 10 % 포르말린 10 ml의 빙초산을 5 ml), 5 분. 직경 ≥ 0.5 mm의 보조 사이트에서 종양 결절을 정의 전이로 결절을 결정합니다. 주 : 종양에 의한 기관지 및 세기관지 막힘, 종양 결절이 15 % 먹물을 흡수 할 수없고, 따라서 정상적인 폐 조직이 검게 될 것이고, 종양 결절이 백색 7 인해 것이다. 이것은 개인의 종양 결절 (그림 2B)의 시각화 수 있습니다. </li></ol> 마우스 배아에서 마우스 배아 섬유 아 세포 (MEFs에를) 준비 (3) <ol><li> 아이소 플루 란 (99.9 %)이 임신 한 쥐를 희생하고 무균 환경을 보장하기 위해 75 %의 에탄올을 스프레이. </li><li> 가위로 목 요도 구멍의 영역에서 피부를 열고 자궁에서 배아를 분리. </li><li> 피부를 열 때 배아를 방해하지 않도록주의하십시오. </li><li> 자궁 뿔을 제거하고 차가운 인트에서 그들을 씻어PBS (PH 7.4)를 화나게. </li><li> 얼음 차가운 PBS (PH 7.4)를 포함하는 10cm 접시에 매우 신중 태반과 장소 배아에서 배아를 분리합니다. </li><li> 배아로부터 헤드, 간, 창자를 제거하고 PBS (PH 7.4)를 함유하는 12 웰 조직 배양 플레이트의 나머지 부분을 배치했다. </li><li> 또 다른 12 웰 플레이트 포함 트립신 EDTA에 배아 체를 전송하고 멸균 면도날과 가위를 사용하여 작은 조각 (~ 1mm 3)로 절단. 37 ℃에서 5 분 동안 트립신 EDTA 5 ㎖와 다진 재료를 품어. </li><li> 다진 배아 1 ml의 우 태아 혈청 (FBS)을 첨가함으로써 트립신을 비활성화한다. 원심 분리기를 10 분 동안 200 XG 속도 현탁액을 조심스럽게 상층 액을 제거 따뜻한 DMEM 배지 1 ㎖로 펠렛을 재현 탁하고 10 DMEM의 ㎖ (20 %의 FBS와 2X 페니실린 / 스트렙토 마이신을 함유 10cm 접시에 모든 내용을 전송 ). </li><li> 이틀 동안 매체 매일을 변경합니다. 문화 원하는 셀 때까지 세포수는 달성된다. </li></ol>

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</ol>

저자는 더 공개가 없습니다.

이 프로토콜은 신선한 마우스의 종양에서 일차 전지를 분리입니다. 이 방법에서 준비 해물 단백질, DNA와 RNA를 분석하는 데 유용합니다. 기본 유방 종양에서 암 세포들은 뇌, 폐, 뼈 1로 전이. 포함 된 전이의 검출을위한 폐를 추출하는 기술이다. 도 2b에 도시 된 바와 같이 잉크 얼룩 폐 염색하여 종양 결절의 시각화를 허용한다. Z-수정 용액에 폐를 고정하는 것은 우리가 폐의 섹션을 확인하고 전이 메커니즘을 연구에 적합한 항체로 염색 할 수 있습니다. 또한 마우스 배아 섬유 아 세포의 분리를위한 프로토콜이 포함되어 있습니다. 이것은 동일한 종류의 동물의 배아 섬유 아세포와 일차 종양 세포의 유전자 발현을 비교하는 데 유용하다. 일차 전지와 MEFs에의 제조에서, 가장 중요한 단계는 재료의 닦지이다. 보라, 적절한 고장없이w 세포 수를 달성 할 것이다. 낮은 세포 카운트가 달성되는 경우, 프로토콜은 여전히​​ 따라야 할 수 있지만 세포 폐암을 추출에서 .. 장기간 배양 할 필요가있을 것이다, 적절한 염색 및 탈색 종양 결절 시각화를 허용하는 것이 필수적이다. 폐에서 RNA / 단백질을 추출하려면, 그것은 흠없는 및 고정되지 않은 있습니다 폐를 사용하는 것이 좋을 것이다. 그러나, RNA는 고정 조직뿐만 아니라 (8)로부터 추출 될 수있다. 유방암의 전이 단계가 영향을받는 개인이 가장 큰 위협이 때문에, 유방 종양의 모든 구성 요소를 가시화하는 것이 중요하다. 유방암 진행의 메커니즘을 이해하는데 중요한 문제는 질병의 진행 9 요점을 되풀이 모델의 유용성이다. 유방 종양 발생 및 전이의 분자 메커니즘의 해명은 생쥐 9, 10, 동물 모델을 이용하여 달성된다. 가장 일반적인 방법은 유도 MOUS전자 유방 종양 유전자, 암 유전자 또는 종양 억제의 돌연변이를 사전 배치 발암 물질을 도입, 매우 침습적 인 암 세포 10-12 주입으로 쥐을 설계하고 있습니다. 획득 후, 상기 종양 유전자 발현을 분석 할 수있다. 이 환자 (13)의 임상 적 결과를 예측 이후 유방암 연구를 위해, 유전자 발현 프로파일이 중요하다. 몇 가지 제한 사항이 유방 종양 연구의 마우스 모델로 존재한다. 마우스는 인간과 분자 및 생리 학적 유사성을 공유하지만, 그들은 항상 인간의 질병 (5)의 모든 측면을 요점을 되풀이하지 않습니다. 특정 유전자 변형 마우스를 사용할 때 고려해야한다. 전반적으로, 마우스 모델은 조직 문화와 고립 된 인간의 종양과 같은 다른 현재 모델에 비해 마우스 유방 종양을 분석하기보다 현실적인 방법을 제공합니다. 함께 찍은,이 프로토콜은 서로 다른 측면의 추가 조사를 위해 수 있습니다유방 종양의.

Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5. 
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse&#8217;s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

<table><tbody><tr><td>DMEM</td><td>HyClone</td><td>SH30243.01</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life&amp;nbsp; technologies</td><td>25300-062</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Collagenase Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Pen Strep</td><td>Life&amp;nbsp; technologies</td><td>15140-122</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gemini Bio-Products</td><td>100-106</td><td>Store at &amp;ndash; 80 &amp;deg;C</td></tr><tr><td>Z-fix</td><td>ANATECH</td><td>#174</td><td>Store at RT</td></tr><tr><td>India Ink</td><td>Yasutomo</td><td></td><td>Store at RT</td></tr><tr><td>AERRANE (Isoflurane)</td><td>Baxter</td><td>NDC 10019-773-60</td><td>Store at RT</td></tr><tr><td>Ethanol</td><td>EMD</td><td>AX0441-3</td><td>Store at RT</td></tr></tbody></table>

primary tumor and mef cell isolation to study lung metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

동물을 희생에서 종양의 진행 단계는 특정 연구 및 IACUC 지침에 따라 달라집니다. 그림 1A, 마우스 유방 종양 바이러스 폴리오 중동 T-항원 (MMTV-PyMT)와 100 일된 C57BL / 6 마우스의 세 개월에 희생되었다. 피부 위 목 요도 오리피스의 영역으로부터 개방하고 피부는도 1b에 나타낸 바와 같이 기관에 쉽게 액세스 할 수 있도록 아래로 고정 하였다. 이는도 1C에서 얻어진 종양에 쉽게 접근 할 수 있었다. 종양에서 얻은 일차 전지는 그림 1D으로 시각화된다. 많은 유전자가 폐 한 유방암의 전이를 매개하기 때문에, 우리는 전이 과정을 연구하기 위해 폐 (도 2A)을 추출 하였다. 폐는 Z-수정과 팽창 종양 결절을 시각화하는 인도 잉크 (그림 2B)로 염색 하였다. 그림 3은 마우스 배아 FIBR를 보여줍니다<html> FVB 변형 마우스 배아에서 분리 oblasts. <img alt="그림 1" src="/files/ftp_upload/52609/52609fig1.jpg" /> 유방 종양에서 일차 전지 1. 준비 그림. (A) 폴리오 중간 T 항원 (PyMT)와 100 일된 암컷 마우스를 희생시켰다. (B) 피부 유방 종양에 쉽게 액세스 할 수 있도록 아래로 고정 하였다. (C) 유방 종양 동물로부터 잘라 내고 . (100 μM 스케일 바) (D) 절연 일차 세포는 둘 베코 변형 이글 중간 10 % 소 태아 혈청 및 항생제가 보충 된 (DMEM)에서 배양 하였다. 이미지는 하루에 3에서 셀을 표시 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> / 52609 / 52609fig2.jpg &quot;를 업로드 /&gt; 그림 2. 마우스 폐 추출 폐 전이를 시각화합니다. (스케일 바 : 500 μm의) (A) 폐는 Z-수정 및 종양 결절을 시각화하기 위해 인도 잉크로 염색 (B)로 팽창했다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52609/52609fig3.jpg" /> . 마우스 배아에서 마우스 배아 섬유 아 세포 (MEFs) 준비 그림 3. MEF 세포 FVB 마우스 배아 (스케일 바 : 100 μm의)에서 추출 하였다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52609/52609fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

차 종양과 폐 전이을 연구하기 위해 MEF 세포 격리

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

primary-tumor-and-mef-cell-isolation-to-study-lung-metastasis

Nanyang Technological University

Research

JoVE Journal

Medicine

15.8K 조회수.  Louisiana State University Health Sciences Center. The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

비디오: Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

폐 종양 세포 모집 분석 결과

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

연구

암 연구학

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

실험 전이 분석

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

의학

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

폐 전이 분석 결과 사용 하 여 다리에 전이성 폐 식민지 공부에서 실용적인 고려 사항

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

순환 종양 세포 시각화 폐 조직에 대 한 폐 식민 분석 결과의 설립

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Acellular과 세포 폐 종양 전이 연구 모델

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

원발성, 악성 흉막 중피종 종양 라인의 생성 및 확장

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

폐 전이로부터의 종양 세포 전파 검사

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

저자 스포트라이트: 유방암 전이를 추적하기 위한 새로운 생체 내 기법을 사용하여 전이에서 전이로의 시딩 해독 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

차 종양과 폐 전이을 연구하기 위해 MEF 세포 격리

요약

더 많은 비디오 탐색

폐 종양 세포 모집 분석 결과

실험 전이 분석

폐 전이 분석 결과 사용 하 여 다리에 전이성 폐 식민지 공부에서 실용적인 고려 사항

순환 종양 세포 시각화 폐 조직에 대 한 폐 식민 분석 결과의 설립

Acellular과 세포 폐 종양 전이 연구 모델

원발성, 악성 흉막 중피종 종양 라인의 생성 및 확장

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

항 종양 특성을 가진 분리 및 호중구의 특성