Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

윤리 선언문 : 모든 동물 실험은 뉴욕 시립 대학의 헌터 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 사전 실험 절차 <ol><li> 동물 시설에 도착하면, 처음에 필터링 상단과 우드 칩 침구와 케이지에 3 BALB / c 마우스를 수용. 쥐가 음식, 퓨리나 설치류 우, 물을 무료로 이용하실 수 있습니다 있는지 확인합니다. 케이지가 일주일에 한 번 청소해야합니다 동안 침구와 음식은 일주일에 두 번 변경해야합니다. </li><li> 분당 1 L의 일정한 유속으로 산소와 함께 기화기 이소 플루 란을 이용하여 마우스의 전신 마취를 유도한다. 이 시스템은 자립하고 정밀 기화기를 포함한다. </li><li> 연결된 정밀 기화기와 가스 / 마취제 시스템을 갖는 플라스틱 상자에 위치 동물. 동물이 시점에서 2 기화기을 절감, 의식이 될 때까지 처음에 2.5 기화기를 설정합니다. </li><li> 이 때, 스위치밸브 라인, 박스로 밀봉하고 노즈콘에 개방. </li><li> 노우즈 콘 (유지 마취)의 설치 위치와 접속하여, 준비 공간에 동물을 이동. </li><li> 마우스 당 주입 5 × 10 6 BNL 1ME A.7R.1 마우스의 간세포 암 세포를 준비합니다. BNL 1ME A.7R.1 매체에서 성장한다 (10 % 열 불 활성화 FBS, 1 % 페니실린 / 스트렙토 마이신 및 2 mM의 글루타민 L) 및 5 %의 공기와 가습 된 배양기에서 37 ℃에서 세포 배양 접시에서 배양 CO 2. </li><li> 주사기 20 G 바늘을 갖는, BALB / c 마우스의 간의 로브로 5 × 106 BNL 1ME A.7R.1 마우스 간암 세포를 함유하는 PBS의 임플란트 100 μL은 차 종양을 생성한다. 착상 후, 집 착상 전 같은 집의 요구 사항에 케이지의 BALB / c 마우스. </li><li> 실험 종료 시점에서, 간세포 암종의 임상 적 증거는 신체 상태의 상당한 감소로서 관찰 할(일반적으로 사주 후 감지) 점수. 이 시점에서, 증착물은 전이성 폐에서 형성 한 주 동안 종양 간에서 개발 한 것을 가능성이있다. 그 후, 종양 세포 순환의 분리 및 전파의 과정을 시작. </li></ol> 종양 세포 분리를 순환 전혈 2. 컬렉션 참고 : 제대로 수술 영역을 소독하고 혼란없는 있는지 확인합니다. 모든 수술 도구를 압력솥 또는 제조업체의 권장 사항에 따라 살균제에 담가 <ol><li> 실험 종료 시점에서 인도적인 안락사 (종양 개발의 임상 적 증거)에 의해 쥐를 희생. 마우스의 Euthanization은 이산화탄소 질식을 포함한다. 마취를 확인하기 위해 적절하게 마우스를 관찰하는 것은 제대로 배포됩니다. 호흡 정지의 원인을 10 분 - 이산화탄소 5 걸쳐 챔버 내로 서서히 방출한다. 심장 내 방혈 및 cervic에 의해 수행알 전위. (5) </li><li> 전체 혈액의 수집을 위해 1 ML의 주사기에 부착 된 25 G 바늘을 사용합니다. 0.01 ㎖의 헤파린 주사기 Heparanize. xyphisternum 약 45 ° 각도로 떨어지는 바늘 즉시 찔린 피부. 심장에 구멍을 천천히 바늘을 전진하고 바늘의 각도를 낮 춥니 다. 꾸준히 장소에 바늘과 주사기를 개최, 500 ㎕를 끌어 - 마음에서 전체 혈액의 1,000 μl를. </li><li> 주사기와 바늘을 제거하고 미리 라벨링 1.5 ML의 발열이없는 튜브에 혈액을 전송합니다. 5 분 동안 1,167 XG에 1.5 ml의 무 발열원 튜브에 수집 전혈을 원심 분리기. 주 : 원심 세 층으로 전체 혈액 샘플을 분리한다 : 적혈구, 버피 코트의 중간 박막과 상부 플라즈마 층으로 이루어진 하부 층 또는 헤마토크리트. </li><li> 피펫 조심스럽게 플라즈마를 제거하고 푸르트에 대해 별도의 1.5 ml의 발열이없는 튜브로 수집휴대없는 핵산 (7)의 검출과 같은 어 실험. </li><li> 순환 성 종양 세포 (CTCS)의 분리를 위해, 적혈구 (RBC)의 용해에 대비 별도의 1.5 ml의 무 발열원 튜브에 버피 코트를 수집한다. 이를 버피 코트 층에 잔류 적혈구를 제거 할 필요가있다. </li></ol> 적혈구 (RBC) 용해 버퍼 3. 레시피 <ol><li> RBC 용해 버퍼의 1 L 솔루션을합니다. </li><li> 염화 암모늄 500 mL 및 트리스 500 mL로한다. 이어서 염화 암모늄 155 mM의 트리스 10 mM의 최종 농도와 적혈구 용해 완충액 1 L의 용액을 만들기 위해 혼합한다. </li><li> 7.5의 pH로 완충 용액. 2-8 ℃에서 적혈구 용해 완충 용액을 저장하고, 직전에 사용하고 RT로 가져온다. </li></ol> 버피 코트 4. 적혈구 용해 <ol><li> 수집 된 버피 코트를 포함하는 무균 무 발열원 1.5 ㎖의 튜브에 적혈구 용해 완충액 1 ㎖를 추가한다. </li><li> 하여 튜브의 내용물을 혼합여러 번 반전. 벤치에 RT에서 5 분 동안 튜브의 내용물을 혼합하여 배양한다. 1,167 X g에서 5 분간 원심 분리하여 혼합 내용. </li><li> 원심 분리 후, 펠릿 백탁이 얻어 질 것이다. 펠릿에 대한 중단하지 않고 차분히 10 % 표백제에 붉은 상층 액을 버린다. 3.5 - 펠릿 백탁 없으면 다시 DO 3.1 단계. 10 RBC 용해 완충액에서 항온 배양 시간을 증가 - 15 분 또한 도움이 될 수있다. </li><li> 10 % 표백제의 생물학적 폐기물의 오염 제거는 24 시간 후 완료되면 안전하게 생물학적 폐기물을 폐기하십시오. </li></ol> 세포 배양 5. 전파 주 : 종양 세포는 전형적으로 세포를 빠르게 접시에 세포를 접종 원래 믹스 풍부 백혈구 증가하고있다. 매체의 반복 변경 CTCS 후속 통로 다음, 모든 백혈구 제거되고 CTCS의 비교적 순수한 인구가 남아있다. <ol><li> 후10 % 표백제로 상등액을 버리고, 세포 배양 물에서 증식을 위해 완전 배양 배지에서 적어도 한 번은 1 ㎖의 인산 완충 식염수 (PBS)에 백탁 펠렛을 세척하고 재현 탁. </li><li> 세척이 완료되면, 펠렛을 완전 BNL 1ME A.7R.1 배지 (FBS 불 활성화 된 10 %의 열, 1 % 페니실린 / 스트렙토 마이신 및 2mM의 글루타민) 1 ㎖를 추가하고 배지에 펠렛을 재현 탁. </li><li> 세포 배양 접시에 세포를 재현 탁 믹스 시드. 공기 및 5 % CO 2의 가습 인큐베이터에서 37 ℃에서 세포 배양 접시를 배양한다. </li><li> 모든 2~3일 세포 배양 배지를 변경한다. 5-7일 후 CTCS는 세포 배양 접시에 부착 및 증식 시작한다. 이 세포들은 25 유로 이상 반복 동결 융해 사이클 (5) 후 가능한 유지됩니다. </li></ol> 간세포 암의 6 검증은 종양 세포 라인을 순환 <ol><li> AP를 수행연쇄 반응 (PCR) 기반 방법 olymerase, 소설 CTC 세포주 원래 주입 BNL 1ME A.7R.1 HCC 라인처럼 마우스 세포 인 확립 확인하는 마우스 β 글로빈 유전자의 단지 하나의 특정 DNA 세그먼트를 증폭. 방법은 원래 기술과 Steube 등에 의해 확인되었다. 5,8 </li><li> 특정 항체를 사용하여 단백질 3 추천 (CREB3L3)를 결합 간세포 특이 마커 캠프 응답 요소에 대한 면역 염색 수행합니다. 이 설립 CTCS가 참으로 원래의 이식 BNL 1ME A.7R.1 세포주처럼 간세포 그 소설을 확인합니다. (5) </li></ol>

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The authors declare that they have no competing financial interests.

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are important in cancer metastasis and have potential for multiple clinical applications, such as for diagnosis3,9,10. This is supported by data from several clinical studies that have showed that CTCs have a strong correlation with the progression of the disease in various cancer types such as pancreatic, prostate and breast, among others3,11,12. Despite the fact that CTCs may be very useful for cancer diagnosis, isolating and characterizing CTCs are challenging because very few are present among billions of blood cells3,7.
A significance of isolating CTCs using our technique is that it provides a reproducible model which can be optimized for potential utility in a clinical setting. This may ultimately lead to the possibility of using this method to replace current biopsy procedures that are more invasive and associated with side effects3,13. Modifications of this method, such as peripheral intravenous blood collection, would be expected to utilize it in a clinical setting. Of course, it is acknowledged that isolating CTCs from humans is a much more challenging endeavor due to general low yield4,14. But, in many cases such as in pancreatic cancer, histo-pathologic or cytological validation using tissue biopsies is fruitless in spite of multiple invasive attempts4. Isolating CTCs has been described as &#8220;liquid biopsy&#8221; or &#8220;live biopsy&#8221; of a tumor which enables noninvasive cancer diagnosis and real-time monitoring of therapeutic response3. This methodology models the isolation of CTCs in a mouse model. Thus far, only one FDA- cleared assay, such as Veridex CellSearch System, has been approved to detect CTCs. In one study, this technology identified CTCs in only 11 of 26 patients. This system is associated with some inefficiency, limited purity and high cost4. Moreover, it lacks the important feature of being able to collect viable CTCs whether they are EpCAM-expressing or not for extensive analyses using multiple in vitro and in vivo methods.
An important feature of the described method is that it preserves the viability of the isolated CTCs3. Therefore, using this method allows for the subsequent extensive downstream analysis of both the molecular and functional characteristics of CTCs using a variety of techniques. 
In conclusion, a methodology for isolating and propagating CTCs that are viable and can be characterized molecularly and functionally is presented. There is potential that this method can be optimized for clinical applications.

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

<table><tbody><tr><td>Heparin Sodium Salt 1 g</td><td>VWR</td><td>89508-852</td><td></td></tr><tr><td>BTX Tube Micro 1.5 ml Clear NS</td><td>VWR</td><td>89511-254</td><td>1.5 ml&amp;nbsp; pyrogen-free eppendorf Tubes</td></tr><tr><td>Needle Sterile Disp BD 25 G x 1 in</td><td>VWR</td><td>BD305125</td><td>25 G Needle</td></tr><tr><td>Slp Tip SRNG 1 ml 200 each per pack</td><td>VWR</td><td>BD309659</td><td>1 ml syringe tip</td></tr><tr><td>Syringe 1 ml leur lok Pk 100</td><td>VWR</td><td>BD309628</td><td>1 ml syringe</td></tr><tr><td>VWR Forceps Tissue 6</td><td>VWR</td><td>82027-446</td><td>Forceps</td></tr><tr><td>Cyromold intrm 15 x 15 x 5 mm PK 100</td><td>VWR</td><td>25608-924</td><td>Cyromold</td></tr><tr><td>Cryo-oct compund 4 oz</td><td>VWR</td><td>25608-930</td><td>Oct compound</td></tr><tr><td>VWR Slide sprfrst 25 x 75 mm PK72</td><td>VWR</td><td>48311-703</td><td>Slides</td></tr><tr><td>VWR Cover Glass #2 22 x 50 mm</td><td>VWR</td><td>48-382-128</td><td>Cover Glass</td></tr><tr><td>VWR Slide Box True North Fm Pu</td><td>VWR</td><td>89140-278</td><td>Slide Box</td></tr><tr><td>Super HT PAP Pen</td><td>VWR</td><td>89427-058</td><td>PAP pen</td></tr><tr><td>Water RNASe and DNAse free 2 L</td><td>VWR</td><td>101454-204</td><td>Nuclease Free Water</td></tr><tr><td>Buffer Tris Ultra Pure Grade 500 g</td><td>VWR</td><td>97061-796</td><td>Tris Buffer</td></tr><tr><td>Ammonium Chloride ACS Grade 2, 5 Kg</td><td>VWR</td><td>97062-048</td><td>Ammonium Chloride Buffer</td></tr><tr><td>Falcon Tissue Culture Dish 60 x 15 mm Style polystyrene</td><td>VWR</td><td>353002</td><td>Tissue Culture Dish</td></tr><tr><td>Clorox Germicidal Bleach, Regular</td><td>VWR</td><td>89501-620</td><td>Clorox Bleach</td></tr><tr><td>PBS, 1x (Phosphate-Buffered Saline) without calcium &amp;amp; magnesium (500 ml)</td><td>Thermo Fischer Scientific</td><td>21-040-CV</td><td>PBS, 1x</td></tr><tr><td>DMEM, 1x&amp;nbsp; with 4.5 g/L glucose &amp;amp; L-glutamine without sodium pyruvate</td><td>Thermo Fischer Scientific</td><td>10-017-CV</td><td>DMEM 1x media for BNL 1ME A.7R.1 cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fischer Scientific</td><td>35-010-CV</td><td>FBS</td></tr><tr><td>Penicillin Streptomycin Solution, 100x</td><td>Thermo Fischer Scientific</td><td>30-002-Cl</td><td>Penicillin Streptomycin</td></tr><tr><td>Sorvall Biofuge pico</td><td>Thermo Fischer Scientific</td><td>75002411</td><td>13,000 rpm Centrifuge</td></tr></tbody></table>

isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

여기서 설명한 방법론은 CTCS가 분리 될 수 있다는 것을 보여 주었다. 마우스는 인도적 실험 엔드 포인트에서 안락사시켰다. 프로세스 관련 이산화탄소 질식, 심장 내 방혈 및 경추 탈구. 과정의 주요 단계를 개략적으로는도 2에 도시되어있다. 내부 - 심장 방혈로 수집 전혈 시료는 전술 프로토콜을 이용하여 처리 하였다. 그 후, 원심 분리 RBC 용해시켰다 버피 코트 층을 결정하는데 사용 하였다. 이 후 PBS로 세척 하였다 백탁 펠렛을 수집하는 또 다른 원심 분리 단계에 이어, 세포 배양에서 전파 완전한 세포 배양 배지에 재현 탁 하였다. 올 수있는 문제가 얻어 펠렛은 색상이 명확하지 않을 수 있습니다. 이를 추가 시간을 행하여 아마도 15 분, 또는 -이 문제는 쉽게 10로 증가시켜 적혈구 용해 완충액에서 배양 시간을 변경함으로써 해결 될 수있다.BNL 1ME A.7R.1의 세포에 비해 세 가지 다른 생쥐는도 1에 도시되어 CTCS에서의 대표적인 이미지는 배양으로 전파된다. 빠른 증식과 형태는 시드 세포가 CTCS 인 경우 식별하는 데 도움이됩니다. 그러나, 단계 5.1에 기재된 예컨대 PCR과 같은 면역 검증 기법 - 5.2 CTC 가능한 라인의 설정을 확인하기 위해 필요하다. <img alt="그림 1" src="/files/ftp_upload/52861/52861fig1.jpg" /> 의 동소 이식 동계 마우스 모델에서 세포 배양에 종양 세포 (CTCS)를 순환 그림 1. 전파 간세포 암 전이. 세포는 BNL 1ME A.7R.1 배양 배지에서 15mm X 60mm 세포 배양 접시에 전파되었다. 증식 종양 세포는 접시에 부착하고 별도의 세 가지 마우스에서 표시됩니다. 종양 세포는 연속적 통로를 통해 실행 가능한 남아있다. CTCOL1MEA081211, CTCOL1MEACTC08291의 위상 대비 이미지1 CTCOLUF2419가 BNL 1ME의 A.7R.1 세포 (40X 배율)에 비해 40 배의 배율 (대물 렌즈)에서 촬영되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52861/52861fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 1" src="/files/ftp_upload/52861/52861fig2.jpg" /> 단리 및 전파하는 마우스 암 모델 시스템에서 종양 세포 순환 절차도 2 회로도. 간암 임상 증거가 신체 상태 점수 상당한 감소로 관찰 한 결과, 전혈은 마우스로부터 수거 하였다. 몇 회 원심, 적혈구 용해, 여러 세척 단계 후, CTCS는 전파 세포 배양 접시에 씨앗을 품고 있었다. 종양 세포의 증식이 며칠 후에 관찰 하였다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52861/52861fig2large.jpg" target="_blank">그 클릭하세요</a>이 그림의 더 큰 버전을 보려면 다시.

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

마우스 종양 모델에서 종양 세포 순환의 분리 및 전파

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K 조회수.  Hunter College of The City University of New York. Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

비디오: Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

대장 암 정위 마우스 모델에서의 순환 종양 세포의 분리

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

연구

암 연구학

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

하류 분자 분석 및 전이성 전위 평가를 위한 순환 종양 세포의 미세 조작

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

마우스 횡문근에서 분리 된 원발성 종양 세포에서 종양 구체 유도 및 치료

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

유전학

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

순환 종양 세포 주: 근본적이고 번역적인 연구를 위한 혁신적인 도구

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

저자 스포트라이트: 고해상도 생체 내 영상 및 MRI를 통합하여 정위 신체 방사선 치료 계획 향상

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

중개 암 연구에서 생체 내 현미경 및 자기 공명 영상의 결합을 위한 Dorsal Skinfold Window Chamber 종양 마우스 모델

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

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.

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 ...

의학

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;환자와 같은&quot;동소 이식 동계 마우스 모델 간세포 암의 전이

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

종양 특이 내피 세포의 분리 및 문화 확장

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

마우스 종양 모델에서 종양 세포 순환의 분리 및 전파

요약

더 많은 비디오 탐색

이 비디오의 챕터

대장 암 정위 마우스 모델에서의 순환 종양 세포의 분리

하류 분자 분석 및 전이성 전위 평가를 위한 순환 종양 세포의 미세 조작

마우스 횡문근에서 분리 된 원발성 종양 세포에서 종양 구체 유도 및 치료

순환 종양 세포 주: 근본적이고 번역적인 연구를 위한 혁신적인 도구

중개 암 연구에서 생체 내 현미경 및 자기 공명 영상의 결합을 위한 Dorsal Skinfold Window Chamber 종양 마우스 모델

중개 암 연구에서 생체 내 현미경 및 자기 공명 영상의 결합을 위한 Dorsal Skinfold Window Chamber 종양 마우스 모델

중개 암 연구에서 생체 내 현미경 및 자기 공명 영상의 결합을 위한 Dorsal Skinfold Window Chamber 종양 마우스 모델

차 종양과 폐 전이을 연구하기 위해 MEF 세포 격리

"환자와 같은"동소 이식 동계 마우스 모델 간세포 암의 전이

종양 특이 내피 세포의 분리 및 문화 확장