저자는 어떤 승인이 없습니다.

실험 절차 및 동물의 윤리적 인 대우는 피츠버그 IACUC 대학 (프로토콜 # 1104674)에 의해 승인되었다. G6PD 염색 혼합물 1. 준비 참고 : 다음과 같이 각 시약의 최종 농도가 있는지 확인; 5 mM의 글루코스 -6- 포스페이트 (G6P); 2 mM의 NADP, 5 밀리미터의 MgCl 2, 0.35 mM의 1- 메 ​​톡시 -5- methylphenazinium 메틸 설페이트 (MMPMS) 및 0.8 mM의 NBT. 초기 농도가 40 ㎖의 최종 부피에 대해 유도 된 각 시약 1,8,9. 솔루션 갓 준비하고 매일 사용되었다. <ol><li> 50 ML 튜브에 10월 매체의 35 ml의 200 mM의 pH 7.4의 인산염 완충액 (PB) 2 ㎖를 추가하고 격렬하게 흔들어. 이 솔루션은 ~ pH가 7.4 인 산도 용지 확인합니다. 그렇지 않으면하는 솔루션을 버린다. </li><li> 61 mg을 200 mM의 G6P PB 0.94 ㎖에 용해하고, 추가 61 mg을 200 mM의 NADP PB 0.94 ㎖에 용해시키고, 41 mg의 0.96의 MgCl 2 ml의 100 mM이 PB에 용해시켰다. pH가 있음을 재확인 ~ 7.4 재치시간의 pH 종이. 하지 ~ 7.4 경우, 솔루션을 버린다. </li><li> 200 mM의 PB (PH 7.4) 0.25 ㎖에 녹이고 MMPMS 5 mg을 추가합니다. 적극적으로 분명 어두운 빨간색 균질을 생성하기 위해 생성 된 반응 혼합물을 흔들어. 용액이 혼탁 한 경우, 혼합물은 폐기한다. 반응 혼합물을 포함하는 튜브는 빛에 대한 노출을 최소화하기 위해 호일로 래핑된다. 최종 요소,​​ NBT가 준비되는 동안 실온에서 반응 혼합물을 저장한다. </li><li> O 링 뚜껑을 2 ㎖의 튜브에 NBT 164 mg을 0.4 ml의 무수 디메틸 포름 아미드 (DMF) 및 0.4 ml의 무수 에탄올을 첨가함으로써 별도로 5mM의 NBT 용액을 제조 하였다. 뚜껑을 느슨하게 닫고 (밀봉되지 않은) 및 NBT 용액이 될 때까지 오일 배스에서 격렬한 비등으로 용액을 가져온다. (8) </li><li> 가용화 된 NBT는 RT로 냉각 한 후 OCT를 반응 혼합물에 모든 용액을 추가하고 투명한 용액을 수득 할 수 있도록 격렬히 흔들어. 어두운 빨간색 또는 PURP에 초기 오렌지 - 레드의 솔루션 색 변화를 관찰시간이 지남에 따라 제작 색상. 이것은 정상입니다. </li><li> 사용하기 전에 1 시간 동안 37 ℃로 따뜻한 방에서 최종 G6PD 염색 혼합물을 놓습니다. </li></ol> 2. 동물 치료 및 조직 컬렉션 <ol><li> DNA 손상 에이전트, 예를 들어, 6 주령 C57BL / 6 수컷 마우스 치료, IP 주입하여 200 μL PBS의 볼륨에서 10 ㎎ / ㎏ 아족시 메탄 (AOM). IP 주입하여 PBS 200 μL와 제어 쥐를 치료​​. </li><li> 구십일에서 공동으로 IACUC 프로토콜에 의해 승인 된 자궁 경부 전위 다음 2 질식을 쥐를 안락사. </li><li> 수술로 바로 흉골의 기지로 항문 위에서 복강을 엽니 다. 체강에서 소장을 이동하지만, 그것을 절제하지 않습니다. 그냥 직장에 맹장 아래의 하부 대장을 절제. (10) </li><li> 조심스럽게 마이크로 해부 가위를 사용하여 대장의 한 측면을 따라 슬라이스하고 깨끗한 멸균 PBS로 세척하여 열린 대장에서 대변을 제거합니다. 소비세 스위스 롤조직 금형의 내강 바깥 측면 및 장소 D 결장. (10) </li><li> 완전히 듀어는 액체 N 2를 포함에 10월 매체와 장소 금형에 콜론을 담그지. 플라스틱 기재는 단지 간섭 단층 매체가 얼어 때까지 액체 N 2와 접촉하도록 몰드를 잡아. </li><li> 동결 절편을 절단 할 때까지 -80에서 냉동 조직 블록을 저장합니다. G6PD 효소의 불 활성화 될 것입니다 어떤 정착액을 사용하지 마십시오. </li></ol> 냉동 섹션 3. 준비 <ol><li> -25 ℃에서 저온 유지 장치를 이용하여 7 μm의 두께에서 동결 조직 절편을 잘라. 4 연속 7 μm의 섹션을 잘라와 같은 슬라이드 (그림 1)에 그 두 가지를 배치합니다. 콜론 (그림 1)의 한 단계를 표현하기 위해 2 슬라이드 또는 4 연속 7 μm의 섹션을 준비합니다. </li><li> 홍보의 마지막 부분에서&gt; 50 ㎛의 최소 거리와 다음 단계에 대해 반복evious 수준. 이 거리는 각각의 레벨에 대해 평가 소낭이 서로 중복되지 않는다고 보장. </li><li> 자주 절단하는 동안 축적 OCT의 잔류 물을 제거하기 위해 100 % 에탄올로 그라 이오 스탯 블레이드를 닦아냅니다. 단면 처리가 다시 시작되기 전에, 건조 블레이드는 축적 된 정전기를 소산하는 대전 드라이어 시트로 닦는다. C. O -80에서 냉동 섹션 스토어 슬라이드 </li></ol> 동결 절편 조직 4. 염색 <ol><li> 습도가 높은 37 ℃로 따뜻한 방에서 효소 조직 화학 반응을 수행합니다. 참고 : 배양 오븐 또는 슬라이드 따뜻한의 효소 반응을 시도 가난과 일치하지 G6PD 염색되었다. </li><li> 실리콘 그리스 (그림 1)를 사용하여 서로 결합 된 두 개의 1.5 cm X 0.15 cm 스틸 와셔 (내부 직경 x 높이)를 사용하여 조직 염색을위한 우물을 구축 할 수 있습니다. 센터와 잘의 기본에 맞게 파라 필름을 잘라 잘라 잘 장소슬라이드에 조직 섹션을 통해 D. 참고 : 세탁기의 바닥에있는 파라 필름은 슬라이드와 조직 섹션에 점성 G6PD 염색 혼합물을 유지 와셔 사이에 실을 만듭니다. 이 연구에서 각 조직 섹션은 G6PD 반응 염색 혼합물의 동일한 볼륨을받을 수 있도록 0.5 ml의 볼륨 -이 구조는 0.4로 잘 있습니다. </li><li> 10 분 동안 따뜻한 방에서 37 O C에서 냉동 조직 슬라이드를 품어. 37 ℃에서 45-50 분 동안 각 조직 절편 웰 (슬라이드 당 2 부)에 G6PD 염색 반응 혼합물을 추가한다. 따뜻한 방에서 슬라이드를 제거하고 조심스럽게 슬라이드 오프 스테인레스 스틸 우물을 들어 올립니다. </li><li> 긴 가장자리에 설정 슬라이드는 반응 매체가 배출되도록합니다. 30 ~ 60 분 동안 100 mM의 PB (PH 7.4)에 넣어 슬라이드 조직 염색을 방해하지 않고 조직에서 G6PD 염색 반응 혼합물을 제거합니다. PB 염을 제거하기 위해 5 ~ 10 분 동안 증류수에 슬라이드를 잠수함 </li><li> 추가로 조직을 밀봉조직에 적기에서 플루오로 젤을 보내고하고 건조를위한 ~ 30 분 대기. 돌연변이 지하실의 식별을 혼동 할 수 있습니다 거품을 피하십시오. 주 : 기포가 형성되면, 절차는 증류수에 담가 플루오 겔의 제거 후에 반복 될 수있다. 그들이 트랩 기포 경향이 커버 전표를 사용하지 마십시오. </li></ol> G6PD 돌연변이 납골당과 돌연변이 주파수 5. 결​​정 <ol><li> 광학 현미경 G6PD 돌연변이 지하실에 대한 슬라이드를 분석합니다. 주 : 완전 돌연변이 토굴을 식별하는 데 사용 기준은 (ⅰ) 전체 토굴 푸른 색의 결여였다 (ⅱ) 토굴의 외부 구조 (III) 돌연변이는 굴 (IV)의 두 관찰자 돌연변이 같은 토굴을 확인했다, 7 μm의 인접 부분에서 관찰되었고, 그대로였다. 인접 7 μm의 섹션에서 관찰 돌연변이는 하나의 돌연변이로 계산됩니다. </li><li> 이미지 각 G6PD 돌연변이 40 배 배율에서 crypt와는 5.0 메가 픽셀 카메라를 사용하여 이미지를 카탈로그이미징 소프트웨어. </li><li> 배율 10 배 (도 1C)에서의 최소 표면적과 이미지를 가진 레벨에서 각 마우스의 대표 부분을 선택하여 마우스에서 조사 소낭의 총 수를 정량화. 참고 : 수준은 그들의 근접에 종종 하나 이상의 섹션에 지하실을 돌연변이 보여 연속 4 7 μm의 섹션 (그림 1D)를 나타냅니다. 레벨은 대장의 다른 지역에서 지하실을 시각화 할 수 μm의 50&gt;로 구분됩니다. </li><li> 각각의 이미지 파일을 열고 시각 (도 1C에서 상자로 표시됨) 파일을 분석하여 레벨 소낭의 개수를 카운트. 이미지 파일을 하나의 파일에 낮은 300&gt;에 20 지하실에서 관찰 된 지하실의 수에 따라 달라질 수 있습니다. </li><li> 각 콜론 G6PD 돌연변이 지하실 스크리닝 수준의 숫자로 각 마우스에 대해 선택된 레벨 (도 1C)에서 소낭의 총 수를 곱한다. 예를 들면, 총 횟수돌연변이 지하실에 대한 검사 8 단계를 곱한 선택한 레벨 당 1,250 지하실 10,000 지하실의 총 수와 동일합니다. 통계 분석을 위해 각 대장 10,000 지하실의 최소를 평가합니다. </li><li> 각각의 치료를위한 MF를 계산하는 스크리닝 소낭의 전체 숫자로 모든 동물에서 관찰 된 돌연변이의 수를 조합. 처리되지 않은 쥐의 배경 줄기 세포 MF 수준은 &lt;0.1x10 -4 이전 C57BL / 6 마우스에보고에 가깝다 (표 1). 11 </li></ol>

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The authors have nothing to disclose.

종종 유전자 독성 화합물의 효과는 DNA를 수정하는 능력에 의해 결정된다. 이 작업은 일반적으로 조직을 분리하고 DNA 부가 물의 글로벌 레벨을 측정하여 수행됩니다. AOM, 이것은 결장 O 6 -methylguanine 부가 정량 포함한다. 이러한 줄기 세포의 틈새와 같은 특정 유형의 세포 내에서의 손상에 대한 이러한 접근 정보를 이용하여, 손실된다. 부가 물 중 일부분 결국 고정 돌연변이로 변환되기 때?...

대장 암은 DNA를 손상시킬 수 환경 에이전트 및식이 구성 요소에 대한 노출을 포함하고 (예를 들어 APC)를 종양 억제 유전자에 돌연변이를 암 유전자에 체세포 돌연변이를 활성화 (예를 들어, 베타 카테닌) 또는 비활성화 생산하는 것으로 생각됩니다. 그러나, 이들 임계 돌연변이 결장 줄기 세포에서 발생하는 것으로 가정한다. 인해 대장 상피의 고유 토굴 아키텍처, 이는 대장 암 발생과 관련된 화학 물질에 동물을 노출 한 후 결장 줄기 세포 돌연변이를 측정 할 수있다. 돌연변이가 토굴 내의 모든 세포에 존재하므로 여러 X-연결 효소는, 줄기 세포에서 발생하는 돌연변이에 대한 지표 역할을 할 수 있습니다. 이전에, 절차는 마우스에서 결장 종양을 유도 화학 또한 X 결합 글루코오스 -6- 인산 탈수소 효소 (G6PD) 유전자 돌연변이 분석을 통하여 대장 체세포 줄기 세포 돌연변이를 생성하는 것이 입증 된 문서. 1-3방법은 임의의 선택 압력없이 결장 줄기 세포 무작위 체세포 돌연변이의 빈도를 정량화. 절차는 처리 및 제어 생쥐로부터 결장 냉동 미 정착 부의 생산 G6PD 기능적 활성을 생산 세포의 결여 소낭의 식별을 포함한다. , 흰색으로 보이는 이러한 돌연변이 지하실은 돌연변이는 돌연변이 G6PD 유전자를 품고 자손 초래했다 줄기 세포에서 발생했음을 나타냅니다. 효소 분석에서, G6PD 결핍 돌연변이 소낭은 니트로 블루 테트라 졸륨 (NBT)의 환원에 필요한 글루코오스 -6- 인산을 산화 할 수 없다. G6PD 효소가 작동하는 경우, 염색 액에 NBT는 효소 및 청색 &quot;염색&quot;셀의 위치에서 축적 불용성 포르 마잔과 침전물로 감소된다. G6PD 돌연변이있는 세포는 푸른 스테인드 야생형 지하실에 대비 희끄무레 남아있다. 이 방법은 &quot;NULL&quot;표현형이 돌연변이를 측정한다. EN 때문에하는 효소, 예컨대 G6PD 같이 섹션이 수용액에 배치되는 경우 비 고정 조직 절편에 세포 밖으로 확산 될 수있는, 그것은 분석 될 수있는 조직 절편에서 효소를 안정화 할 필요가있다. (4)에서의 효소의 안정성 조직은 G6PD 효소 반응에 필요한 시약 작은 분자의 확산을 방해하지 않아야합니다. 우리는 원래의 절차에 큰 변화의 숫자를 만들었습니다. 중요한 효소 염색 배지 pH 조절 및 상기 분석에 사용 된 성분의 용해를 용이하게 최적의 절단 온도 화합물 OCT (), 폴리 비닐 알코올로부터 변경되었다. 각 조직 섹션 G6PD 반응 혼합물을 동일한 용적을 수신하도록 강철 와셔 이루어지는 0.5 ml를 웰​​ - 염색 0.4을 사용하여 수행된다. 절차없이 결장 조직의 큰 표본을 제공 묘화 부에 소낭의 수를 추정하기 위해 개발 된수동&gt; 각 대장 10 (5) 지하실을 계산합니다. 7 μm의 인접 조직 절편의 분석은 잠재적 염색 아티팩트를 감소 하나 이상의 슬라이드에 토굴 돌연변이의 시각화를 제공한다. 이러한 변경 절차가보다 효율적이고 재현합니다. 이 수정 된 프로토콜을 사용하여, 우리는 설치류 공지 결장 발암 아족시 메탄을 위해 C57BL / 6 마우스의 결장 노출 후 줄기 세포의 돌연변이 빈도를 정량화 하였다. 5-7

<table><tbody><tr><td>reagents</td><td></td><td></td><td></td></tr><tr><td>nitroblue tetrazolium</td><td></td><td></td><td></td></tr><tr><td>NADP</td><td></td><td></td><td></td></tr><tr><td>glucose-6-phosphate</td><td></td><td></td><td></td></tr><tr><td>1-methoxy-5-methylphenazinium methyl sulfate</td><td></td><td></td><td></td></tr><tr><td>dimethyl formamide</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer (pH 7.4</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>light microscope equipped with 5 megapixel camera</td><td></td><td></td><td></td></tr><tr><td>cryostat</td><td></td><td></td><td></td></tr><tr><td>warm room&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

quantification of  colonic stem cell mutations

줄기 세포의 돌연변이를 측정 할 수있는 능력은, 화학 물질은 잠재적으로 암을 초래할 돌연변이를 유도 할 수 있다면, 어느 정도에 중요한 세포 집단의 정량화 할 수있는 강력한 도구이다. 상기 X 연결된 글루코스 -6- 포스페이트 탈수소 효소 유전자의 줄기 세포 돌연변이를 정량화하기 위해, 효소 분석의 사용은 이전에보고 된 바있다. (1)이 방법을 산출하는 반응 혼합물과 절개 된 조직의 동결 절편과 배양의 준비가 필요 세포가 기능적 글루코오스 -6- 인산 탈수소 효소 (G6PD) 효소를 생산하는 경우 청색. 그렇지 않은 경우, 셀은 하얗게. 우리는 폴리 비닐 알코올 대신에, 최적의 절단 온도 화합물 OCT () 매체를 이용하여 반응 혼합물을 수정했다. 이는 pH 측정을 용이 G6PD 염색 성분 가용화를 증가시키고 G6PD 효소의 확산을 제한한다. 돌연변이가 줄기 세포에서 발생 함을 입증하기에, 전체 토굴은 G6PD 효소가 부족합니다활동. 줄기 세포 비호에만 G6PD 표현형 돌연변이 토굴에서 자손 모두 G6PD 효소 활성이 결여된다. 줄기 세포의 변이를 식별하는 지하실, 연속 4 인접 동결 절편 (레벨) 7 μm의 두께로 절단 하였다. 인접 컷의 제조 방법은이 같은 변이 토굴 인접한 부분에서 관찰되기 때문에 토굴 완전히 돌연변이 된 형태를 제공한다. 50 ㎛의 간격이었다 조직 샘플과 슬라이드는 마우스 당&gt; 10 4 지하실의 합계를 평가 제조 하였다. 돌연변이 빈도는 치료 그룹의 야생형 (청색) 소낭의 숫자로 ÷ 관측 돌연변이 (흰색) 소낭의 수이다.

동물에 결장 줄기 세포 돌연변이를 측정 할 수있는 능력은 암 유발에 돌연변이를 상관하는 유일한 방법을 제공한다. 일반적으로, 그것은 암화 과정에서 중요한 단계는 종양 유전자 돌연변이의 활성화 및 / 또는 종양 억제 유전자를 불 활성화 돌연변이가 포함한다고 가정한다. 우리는 PBS 200 μL 또는 PBS 200 μL 10 ㎎ / ㎏ AOM과 C57BL / 6 마​​우스를 주입. AOM은 공지 결장 발암 물질이다. 콜론은 줄기 세...

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

대장 줄기 세포 돌연변이의 정량화

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

quantification-of-colonic-stem-cell-mutations

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.6K 조회수.  University of Pittsburgh. We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

비디오: Quantification of  Colonic Stem Cell Mutations

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

터치 인쇄물 세포학을 사용 하 여 병 적인 임상 표본에서 높은-품질 종양 DNA를 간단 하 고 빠른 방법

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

연구

암 연구학

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

항체 Microarray 및 형광 멀티플렉싱을 사용하여 대장암 세포 표면 단백질 프로파일

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

면역학 및 감염병학

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

대변 DNA 무결성 검출에 의한 대장암 위험 및 유병률 평가

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

대장 HT29 유래 암 줄기 와 같은 종양스피어에서 드라이버 유전자의 발견

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

정제 된 조혈 줄기 / 전구 세포에 DNA 돌연변이를 확인

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

표적 시퀀싱 접근법을 통한 비교 병변 분석

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

행동분석학

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및<em&gt; 예 생체 내</em&gt; 섬유 아세포

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

의학

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

결함 Pms2, ERCC1, Ku86, 결장암을 진행하는 동안 필드 결함에 CcOI

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

생물학

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and &#946;-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 &plusmn; 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops ...