저자가 공개하는 게 없다.

PaaS를위한 유리 Coverslips는 1. 준비 <ol><li> 낮은 린트 물티슈 청소 12mm의 된 커버. </li><li> 불꽃 12mm 커버 슬립과 핀셋을 사용하여 분젠 버너 불꽃을 통해 전달하여 각 35mm의 유리 바닥 접시의 마이크로 웰의 14mm coverslip에. </li><li> 실온에서 5 분 동안 0.1 N NaOH를 200 μL로 마이크로 웰 치료. </li><li> 공기 대기음을 30 분 동안 마이크로 웰을 건조. </li><li> 흄 후드에서 실온에서 10 분 동안 3- 아미노 프로필 트리 메 톡시 실란의 50 내지 100 μL로 마이크로 웰 치료. 이 화학 물질은 플라스틱과 반응; 따라서, 유리 피펫을 사용하여 플라스틱 접시와의 접촉을 피하기 위해 마이크로 웰 완전히 채워지지 않는다. </li><li> 3 아미노 프로필 트리 메 톡시 실란이 명확해질 때까지 약 10 분 동안 초순수와 마이크로 웰을 세척 할 것. </li><li> 초순수 두 번 스퀴즈 병을 사용와 마이크로 웰을 씻어. </li><li> 초순수의 2 ml로 마이크로 웰을 세척10 분 동안 중간 속도 (~ 1 Hz로) 설정에 로커 t 실온. </li><li> 공기 대기음을 30 분 동안 마이크로 웰을 건조. </li><li> 30 분 동안 중간 속도 (~ 1 Hz로) 설정 로커에 실온에서 0.5 % 글루 타르 알데히드 용액 2 ㎖와 함께 마이크로 웰 치료. </li><li> 10 분 동안 중간 속도 (~ 1 Hz에서) 설정 로커에 실온에서 초순수 2 ㎖와 마이크로 웰을 세척 할 것. 30 분의 총 두 번 더 반복한다. </li><li> 30 분 동안 가파른 각도 (60 ° 이상)에서 건조 마이크로 웰. 참고 : 요리는 데시 케이 터 2 개월 동안 저장할 수 있습니다. </li></ol> Invadopodia 분석 실험을위한 PaaS를 2. 준비 <ol><li> 소프트 PAA (8 %의 아크릴 아미드 및 0.05 % BIS)의 1 ㎖의 용액의 경우, 40 % 아크릴 아미드 200 μL, 2 %의 BIS 25 μL, 및 초순수 574 μL (표 1)를 결합한다. </li><li> 하드 PAA (8 %의 아크릴 아미드 및 0.35 % BIS)의 1 ㎖의 용액의 경우, 40 %의 아크릴 아미드, 175 # 200 μL를 결합181; L ​​2 %의 BIS, 및 초순수 409 μL (표 1). </li><li> 강성 PAA (12 % 아크릴 아미드 및 0.60 % BIS)의 1 ㎖의 용액의 경우, 40 % 아크릴 아미드 300 μL, 2 % BIS 300 μL, 및 초순수 169 μL (표 1)를 결합한다. </li><li> 15 분 동안 용액을 탈기시킨다. 각각, 부드러운 하드, 단단한 PAA 솔루션에 초순수에 1 ㎎ / ㎖ 인간의 플라즈마 피브로넥틴의 200, 215, 230 μl를 추가합니다. 모든 용액의 경우, 10 ㎎ / ㎖ 아크릴산 N 히드 록시 숙신이 미드 (NHS) 에스테르 1 μL, 100 ㎎ / ㎖의 암모늄 퍼 설페이트 5 μL, 및 N, N, N &#39;, N&#39;- 테트라 메틸 에틸렌 디아민 (TEMED 2 μl를 추가 ; 표 1). 부드러운 피펫으로 혼합 및 솔루션에 거품을 만들지 마십시오. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA (1 ml)에 </td><td> 40 % AA (μL) </td><td> 2 % (μL) </td><td> 초순수 물 (μL) </td><td> 1 ㎎ / ㎖ FN (μL) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 ㎎ / ㎖ NHS 에스테르 (μL) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 ㎎ / ㎖ APS (μL) </td><td> TEMED (μL) </td><td> 탄력있는 계수 (PA) </td></tr><tr><td> 부드러운 </td><td> (200) </td><td> (25) </td><td> (574) </td><td> (200) </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 1,023 </td></tr><tr><td> 단단한 </td><td> (200) </td><td> (175) </td><td> (409) </td><td> (215) </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 7307 </td></tr><tr><td> 엄격한 </td><td> (300) </td><td> (300) </td><td> 169 </td><td> (230) </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 22692 </td></tr></tbody></table> 표 1 : 성분 voluMES 및 invadopodia 분석법에 사용 부드럽고 단단한 경질 PAAS 위해 탄성률 얻어진. AA = 아크릴 아미드, FN은 피브로넥틴, APS는과 황산 암모늄 =를 =. <ol start="5"><li> 각 마이크로 웰의 중심에 PAA 용액 8.48 μL 피펫. 참고 :이 볼륨은 이론적으로 두께 75 ㎛의 PAA를 산출한다. </li><li> 조심스럽게 핀셋을 사용하여 마이크로 웰의 물방울에에 12mm 커버 슬립의 골조면을 낮 춥니 다. </li><li> 샌드위치 PAA 솔루션은 15 ~ 30 분 동안 중합 할 수 있습니다. 남은 솔루션을 확인하여 중합을 확인합니다. </li><li> 각각의 접시에 1X PBS 2 ㎖를 추가하고 핀셋으로 12mm 된 커버를 제거합니다. 참고 : 10 배 PBS는 실험실에서 만들어 0.011 M KH 2 PO 4, 1.54 M의 NaCl, 0.056 M 나 2 HPO 4로 구성되어있다. </li><li> 5 분 동안 실온에서 1X PBS로 2 ㎖ 마이크로 웰을 세척한다. 15 분의 총 두 번 더 반복한다. </li></ol> 3. P견인 힘 분석 실험을위한 PaaS를 배상 <ol><li> 소프트 PAA (8 %의 아크릴 아미드 및 0.05 % BIS)의 1 ㎖의 용액의 경우, 40 % 아크릴 아미드 200 μL, 2 %의 BIS 25 μL, 및 초순수 566 μL (표 2)를 결합한다. </li><li> 하드 PAA (8 %의 아크릴 아미드 및 0.35 % BIS)의 1 ㎖의 용액의 경우, 40 % 아크릴 아미드 200 μL, 2 % BIS 175 μL, 및 초순수 401 μL (표 2)를 결합한다. </li><li> 강성 PAA (12 % 아크릴 아미드 및 0.60 % BIS)의 1 ㎖의 용액의 경우, 40 % 아크릴 아미드 300 μL, 2 % BIS 300 μL, 및 초순수 161 μL (표 2)를 결합한다. </li><li> 15 분 동안 용액을 탈기시킨다. 각각, 부드러운 하드, 단단한 PAA 솔루션 1 ㎎ / ㎖ 인간의 플라즈마 피브로넥틴의 200, 215, 230 μl를 추가합니다. 초음파 처리 30 초 200 nm의 적색 형광 마이크로 스피어 (605분의 580 나노 미터의 여기 / 방출)의 8 μL. 각 솔루션의 경우, 200 nm의 적색 형광 마이크로 스피어의 8 μl를 추가10 ㎎ / ㎖ 아크릴산 NHS 에스테르 1 μL, 100 ㎎ / ㎖의 암모늄 퍼 설페이트 5 μL, TEMED 및 (표 2)의 2 μL. 부드러운 피펫으로 혼합 및 솔루션에 거품을 만들지 마십시오. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA (1 ml)에 </td><td> 40 % AA (μL) </td><td> 2 % BIS (μL) </td><td style="padding-left: 4px; padding-right: 4px;"> 초순수 물 (μL) </td><td> 1 ㎎ / ㎖ FN (μL) </td><td> 마이크로 분야 (μL) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 ㎎ / ㎖ NHS 에스테르 (μL) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 ㎎ / ㎖ APS (μL) </td><td> TEMED (μL) </td><td> 탄력있는 계수 (PA) </td></tr><tr><td> 부드러운 </td><td> (200) </td><td> (25) </td><td> (566) </td><td> (200) </td><td> 8 </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 1,023 </td></tr><tr><td> 단단한 </td><td> (200) </td><td> (175) </td><td> (401) </td><td> (215) </td><td> 8 </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 7307 </td></tr><tr><td> 엄격한 </td><td> (300) </td><td> (300) </td><td> (161) </td><td> (230) </td><td> 8 </td><td> (1) </td><td> (5) </td><td> 이 </td><td> 22692 </td></tr></tbody></table> 표 2 :. 성분 볼륨과 견인력 세이에 사용 부드럽고 단단한 경질 PAAS 위해 탄성률 얻어진 AA = 아크릴 아미드, FN은 피브로넥틴, APS는과 황산 암모늄 =를 =. <ol start="5"><li> 각 마이크로 웰의 중심에 PAA 용액 8.48 μL 피펫. 참고 :이 볼륨은 이론적으로 두께 75 ㎛의 PAA를 산출한다. </li><li> 조심스럽게 일의 골조 측면을 낮출핀셋을 사용하여 마이크로 웰의 물방울에에 전자 12mm coverslip에. </li><li> 샌드위치 PAA 솔루션은 15 ~ 30 분 동안 중합 할 수 있습니다. 남은 솔루션을 확인하여 중합을 확인합니다. </li><li> 각각의 접시에 1X PBS 2 ㎖를 추가하고 핀셋으로 12mm 된 커버를 제거합니다. </li><li> 5 분 동안 실온에서 1X PBS로 2 ㎖ 마이크로 웰을 세척한다. 15 분의 총 두 번 더 반복한다. </li></ol> Invadopodia 분석 실험을위한 ECM 4. 준비 <ol><li> 37 ° C에 젤라틴 용액 (1 % 젤라틴 1 % 자당)를 가열한다. </li><li> 45 °의 각도로 유리 바닥 요리를 진행할 때 마이크로 웰의 바닥으로부터 신중 흡 인물을 1 분 동안 젤라틴 용액 150 μL로 PAAS 치료. </li><li> 60 분 동안 건조를 최적화하기 위해 가파른 각도 (60 ° 이상)에서 마이크로 웰의 PAAS에 젤라틴의 나머지 얇은 층을 건조. </li><li> i에서 냉장 0.5 % 글루 타르 알데히드 용액 2 ㎖와 마이크로 웰 치료30 분 동안 실온에서 15 분 동안이어서 CE. </li><li> 5 분 1X PBS 2 ㎖와 마이크로 웰을 세척 할 것. 15 분의 총 두 번 더 반복한다. </li><li> 1 분 동안 수소화 붕소 나트륨 용액 2 ml로 마이크로 웰 치료. 조심스럽게 젤라틴의 표면에 형성하는 거품을 제거하기 위해 벤치에 요리를 누르십시오. </li><li> 대기음을 5 분 동안 1X PBS로 2 ㎖ 마이크로 웰을 씻어. 15 분의 총 두 번 더 반복한다. </li><li> 15 분 동안 4 ° C에서 175,000 XG에 50 μg의의 /의 1X PBS와 ml의 원심 분리기에 FITC 표지 인간의 플라즈마 피브로넥틴 솔루션을 희석. 시판 표시 피브로넥틴를 구입하거나 다음과 같이 레이블 : <ol><li> 투석 튜브의 보레이트 완충액 (170 mM의 나트륨 테트라 보레이트 40 mM의 염화나트륨)과 장소 10ml에 피브로넥틴 5mg을 녹인다. </li><li> 자기 교반기에 실온에서 용해 FITC 6 ㎎을 함유하는 보레이트 완충액 200㎖에 배관을 배치하여 피브로넥틴 레이블어둠 속에서 1.5 시간 동안 가장 낮은 설정에서 설정합니다. </li><li> 두 볼륨이 어두운 추운 방 (4 ° C)에서 3 일 동안 가장 낮은 설정으로 설정 자석 교반기에 1X PBS의 500 ml로 투석은 하루 변경됩니다. </li><li> (1 : 200) 희석 된 분취 량의 광학 농도에 기초하여 계산 FITC 표지 된 피브로넥틴 농도가 280 nm 내지 493 nm에서의 AS는 [OD 280 - (0.36 X OD 493)] / 1.4 배 (200)의 2 배의 희석 인자는 (있는 계정 ㎎ / ㎖ 단위로 얻어진 다음 단계에서 피브로넥틴 농축 글리세롤) 미국. </li><li> 4 ° C에서 하룻밤 어둡고 추운 방에서 가장 낮은 설정으로 설정 자석 교반기 50 % 글리세롤 용액에 하룻밤 투석.) </li></ol></li><li> 어둠 속에서 60 분 동안 실온에서 FITC 표지 된 피브로넥틴 용액 150 ㎕를 마이크로 웰과 치료. </li><li> 에서 유리 바닥 요리를 들고 때 조심스럽게 마이크로 웰의 바닥에서 FITC 표지 피브로넥틴 솔루션을 대기음45 ° 각도는 다음 멸균 어두운 세포 배양 후드에서 10 분 동안 실온에서 70 % 에탄올 용액으로 유리 바닥 접시를 채운다. 또한 유리 바닥 접시 뚜껑을 채우거나 70 % 에탄올에 배어 낮은 보풀 물티슈로 닦아. </li><li> 5 분 1X PBS로를 작성하여 바닥이 유리로 요리를 씻으십시오. 15 분 총 1X PBS 2 ㎖와 두 번 더 반복합니다. </li></ol> Invadopodia 시험 법 5. 준비 및 이미징 <ol><li> (1 DMEM 및 5 %의 낮은 단백질 혈청, 10 % 소 태아 혈청, 및 새롭게 추가 된 표피 성장 인자 20 ng를 / ㎖와 함께 RPMI 1640 1) 유리 바닥 마성 및 invadopodia 배지 2 ㎖로 25,000 암세포 추가 하룻밤 배양한다. </li><li> 대기음 세포를 고정을 20 분 동안 실온에서 어두운 곳에서 3.7 % 파라 포름 알데히드 용액 2 ㎖와 함께 마이크로 웰을 처리하고. </li><li> 1X PBS 2 ml의 두 빠른 세척 한 후, 방 템피에서 0.1 % 트리톤 X-100 용액 2 ml로 마이크로 웰을 치료5 분 동안 어둠 속에서 rature는 세포를 Permeabilize 하시려면합니다. </li><li> 1X PBS 2 ㎖로 세척 한 후 바로, 3 % 소 혈청 알부민이 60 분을 차단하는 세포에 대한 실온에서 어두운 곳에서 차단 용액으로 마이크로 웰을 치료. </li><li> 60 분 동안 실온에서 어두운 곳에서 차단 용액에서 : 마우스 항 cortactin 항체 (750 1) 150 μL와 마이크로 웰을 인큐베이션. </li><li> 5 분 1X PBS 2 ㎖와 마이크로 웰을 세척 할 것. 15 분의 총 두 번 더 반복한다. </li><li> (500 1) 및 546 팔로이 딘 (1 : 750) 60 분 동안 실온에서 어두운 곳에서 차단 용액에 150 ㎕의 염소 항 - 마우스 항체 633 마이크로 웰을 인큐베이션. </li><li> 5 분 1X PBS 2 ㎖와 마이크로 웰을 세척 할 것. 15 분의 총 두 번 더 반복한다. </li><li> 각각의 마이크로 웰을 작성하여 22 × 22 커버 슬립 커버하는 매체를 장착 여섯 방울을 추가합니다. </li><li> 이미지 cortactin과 액틴이 647분의 632 나노 미터의 여기 및 방출 필터를 사용하여 invadopodia을 확인하고570분의 556 nm의, 각각, 와이드 - 필드 도립 현미경 높은 NA, 40X 오일 침지 렌즈를 사용. 마찬가지로, 화상 피브로넥틴 518분의 492 nm 인 여기 및 방출 필터를 사용하여 ECM 분해 동정을 행 하였다. </li></ol> 6. 준비 및 견인 힘 분석 실험의 영상 <ol><li> 유리 바닥 요리에 invadopodia 매체의 2 ㎖에 15,000 암 세포를 추가하고 밤새 배양한다. </li><li> 유리 바닥 요리 대기음 매체와 같은 보조제 (5 % 낮은 단백질 혈청, 10 % 소 태아 혈청, 및 새롭게 추가 된 표피 성장 인자 20 ng를 / ㎖), 사전에, L-15 배지 2 ㎖로 대체 37 ° C에 -warmed. </li><li> 거꾸로 현미경 1 시간 동안 습도가 높은 37 ° C로 사전 평형에 환경 챔버의 유리 바닥 요리를 품어. </li><li> 휴대 전화 위치를 표시하기 위해 높은 NA, 자동화 단계와 넓은 필드 거꾸로 현미경에 40 배 렌즈를 사용하여 이미지의 네 세트를 가지고 : <ol><li> 에에 이미지 세포위상차 ( &quot;단계&quot;화상)을 사용하여 (P)의 PAAS. </li><li> PAAS 상단의 마이크로 스피어의 제 1 층까지 아래 셀 중심으로 약간의 645분의 560 nm의 여기 및 방사 필터를 사용하여 형광 이미지의 초점이 미세하게된다 (화상 &quot;스트레스&quot;). </li><li> 또한, (마커로서 미소 구체를 사용하여) PAAS의 바닥에 집중하고 ( &quot;바닥&quot;화상)을 화상. 주 :이 이미지의 목적은 대표 결과에 기재된 견인력 계산을위한 각 위치에서의 실제 두께 PAA 얻을 Z 위치를 기록하는 것이다. </li><li> 이러한 이미지가 여러 위치에서 획득 한 후, 부드럽게 세포를 제거하기 위해 10 % 트리톤-X 솔루션의 220 μL에 섞는다. 앞서 설명한 ( &quot;널 (null)&quot;이미지) 등의 각 위치에서 PaaS를 상단의 이미지 미소. </li></ol></li></ol>

<ol>
	<li>Paszek, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer cell. 8, 241-254 (2005).</li><li>Jaalouk, D. E., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+gone+awry.">Mechanotransduction gone awry.</a> Nature. 10, 63-73 (2009).</li><li>Paszek, M. J., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension+mounts:+mechanics+meets+morphogenesis+and+malignancy.">The tension mounts: mechanics meets morphogenesis and malignancy.</a> Journal of mammary gland biology and neoplasia. 9, 325-342 (2004).</li><li>Parekh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+modulation+of+invadopodia+across+a+wide+range+of+rigidities.">Sensing and modulation of invadopodia across a wide range of rigidities.</a> Biophysical. 100, 573-582 (2011).</li><li>Jerrell, R. J., Parekh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+mediate+extracellular+matrix+degradation+by+invadopodia.">Cellular traction stresses mediate extracellular matrix degradation by invadopodia.</a> Acta biomaterialia. 10, 1886-1896 (2014).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7, e32572 (2012).</li><li>Haage, A., Nam, D. H., Ge, X., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase-14+is+a+mechanically+regulated+activator+of+secreted+MMPs+and+invasion.">Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.</a> Biochemical and biophysical research communications. , (2014).</li><li>Haage, A., Schneider, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+contractility+and+extracellular+matrix+stiffness+regulate+matrix+metalloproteinase+activity+in+pancreatic+cancer+cells.">Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.</a> FASEB J. , (2014).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia:+specialized+cell+structures+for+cancer+invasion.">Invadopodia: specialized cell structures for cancer invasion.</a> Clinical &amp; experimental metastasis. 23, 97-105 (2006).</li><li>Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia.">Invadopodia.</a> Curr Biol. 18, R362-364 (2008).</li><li>Bravo-Cordero, J. J., Hodgson, L., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+invasion+and+migration+during+metastasis.">Directed cell invasion and migration during metastasis.</a> Current opinion in cell biology. 24, 277-283 (2012).</li><li>Alexander, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+promotes+invadopodia+activity.">Extracellular matrix rigidity promotes invadopodia activity.</a> Curr Biol. 18, 1295-1299 (2008).</li><li>Barlow, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+breast+cancer+risk+prediction+model+for+women+undergoing+screening+mammography.">Prospective breast cancer risk prediction model for women undergoing screening mammography.</a> Journal of the National Cancer Institute. 98, 1204-1214 (2006).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+absolute+invasive+breast+cancer+risk+in+white+women+with+a+model+that+includes+mammographic+density.">Projecting absolute invasive breast cancer risk in white women with a model that includes mammographic density.</a> Journal of the National Cancer Institute. 98, 1215-1226 (2006).</li><li>Butcher, D. T., Alliston, T., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tense+situation:+forcing+tumour+progression.">A tense situation: forcing tumour progression.</a> Nature reviews. Cancer. 9, 108-122 (2009).</li><li>Zaman, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10889-10894 (2006).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Munevar, S., Wang, Y., Dembo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+of+migrating+normal+and+H-ras+transformed+3T3+fibroblasts.">Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.</a> Biophysical journal. 80, 1744-1757 (2001).</li><li>Rosel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+Rho/ROCK+signaling+in+sarcoma+cells+drives+invasion+and+increased+generation+of+protrusive+forces.">Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.</a> Mol Cancer Res. 6, 1410-1420 (2008).</li><li>Indra, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+correlation+of+mechanical+forces+and+metastatic+capacity.">An in vitro correlation of mechanical forces and metastatic capacity.</a> Phys Biol. 8, 015015 (2011).</li><li>Parekh, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+invasiveness+by+the+physical+extracellular+matrix+environment.">Regulation of cancer invasiveness by the physical extracellular matrix environment.</a> Cell adhesion &amp; migration. 3, 288-292 (2009).</li><li>Weaver, A. M., Page, J. M., Guelcher, S. A., Parekh, A., Coutts, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

이 작품은 상 수 K25CA143412에서 국립 보건원 (NIH)의 국립 암 연구소 (Parekh 씨)에 의해 지원되었다. 우리는 추가 지원이 이비인후과의 부서에서 제공 한 것을 인정하고 싶습니다. 내용은 전적으로 저자의 책임이며 반드시 국립 보건원의 공식 견해를 대변하지 않습니다.

우리는 침습적 수축성 셀룰러 동작과 상관시킬 invadopodia 견인력 분석의 기초로서 사용될 수있다 PAAS을 제조하는 방법을 제시한다. PaaS를 오래 세포에 강성 효과를보고 견인력 18,24,27를 계산하는 데 사용되었지만,이 프로토콜은 매트릭스에 대한 응답으로 침략과 수축 세포 행동의 상관 관계를 같은 경직성와 PaaS를 기반으로 병렬 분석을 개발하는 최초 기계적 특성. 제대로 바닥이 유리로 ?...

종양 관련 ECM의 강성 액토 마이 오신의 수축성 1-3을 증가시켜 악성 동작을 운전에 중요한 요소로 확인되었습니다. 이 효과는 주로 유방암 세포로 입증되었지만, 매트릭스 강성 암의 다른 유형에 중요한 역할을 할 수 있음 종양 강성을 제안 암 4-8 다양한 유래의 세포의 침입 특성을 변경하는 것으로 밝혀졌다. 침습성 마이그레이션 중 가교 결합 조직을 관통하도록, 암세포는 국소 적 ECM 9 저하 테이나 지역화 invadopodia라고도 액틴 풍부한 접착제 돌출부들을 이용한다. Invadopodia 침략 세포의 특징으로 간주되며 암세포 침윤 및 전이 10,11에 연루되어있다. 이전 작업은 매트릭스 강성이 invadopodia 번호를 조절할 수있는 것으로 나타과 미오신 II 활동과 mechanosensitive 단백질 (12)를 통해 ECM 저하 4,12 관련 지을 수 있고 있습니다. 주어진종양 및 암 밀도 공격성 13,14 사이의 상관은, 이러한 결과는 암 세포 액토 마이 오신 수축력 통해 침윤 및 전이를 구동하는 강성 종양 조직에 응답 할 수있는 메커니즘을 제시한다. 시험관 ECM 강성 및 생체 조직 밀도가 암세포의 침습 1,15-17 행동을 조절하는 것으로 나타났다. 액토 마이 오신 수축력이 과정에서 중요하게 나타나는 반면, 전이성 용량이 증가하는 상관 관계 또는 수축력 6,18-20을 감소 여부에 현재 연구 충돌합니다. 또한, 이러한 힘이 직접 invadopodia 활성 여부 (21)를 매개 알려지지 않았다. 우리는 최근에 암 세포의 수축력이 매트릭스 강성에 의존했고, invadopodia 5 ECM 저하의 예측 것을 발견했다. 이러한 결과는 셀룰러 힘 invadopodia 교류를 매개하여 암의 진행에 중요한 역할을 할 수 있음을 시사종양 미세 환경의 기계적 특성에 대응 tivity. 암세포 (5)의 침입 및 수축 특성을 상관하기 위해, 우리는 이전에 의존 강성 invadopodia 4,12,22 활성을 조사하기 위해 사용 된 서로 다른 강성으로 PAAS 생성을위한 프로토콜을 개질. 화학적 PAAS 걸쳐 인간 혈장 피브로넥틴 가교함으로써, 하이드로 겔이 변형 된 세포는 모두 실험 5와 동일한 강성을 경험하도록 invadopodia 및 견인력 분석 모두에 대한 기준으로 이용 될 수있다. invadopodia 내 분석, 피브로넥틴 매트릭스 열화를 검출하기 PAAS에 포개어 ECM을 연결하기위한 젤라틴 천연 결합 도메인을 제공한다. 견인력 내 분석, 피브로넥틴 세포 견인력을 계산하는 데 사용되는 미세 변위를 검출하는 직접적인 세포 부착을위한 리간드를 제공한다. 우리가 하드, 소프트라는 것을이 Methods,정상 및 암 조직 (23)에 대해보고 기계적 특성의 범위에 걸쳐있는 파 5 1023, 7307 및 22692의, 유리 바닥 요리에 바인딩 및 탄성 계수, E가된다 엄격한 PAAS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

리지드 종양 조직 강하게 암 세포 이동 및 침입 조절에 연루되어왔다. 가교 조직을 통해 침략 마이그레이션은 단백질 가수 분해 세포 외 기질 (ECM)을 저하 invadopodia라는 굴지의 풍부한 돌출부에 의해 촉진된다. Invadopodia 활성은 강성 신호 액토 마이 오신 수축력을 통하여 이러한 세포 내 구조를 조절할 수 있음을 시사 ECM 강성 암세포 수축력에 의존하는 것으로 밝혀졌다. 암세포의 침윤성 및 수축 특성은 서로 다른 강성의 폴리 아크릴 아마이드 젤 (PAAS)에 기초 invadopodia 및 견인력 세이를 이용하여 시험 관내에서 상관 될 수있다. 암세포의 침윤성 및 수축 특성은 서로 다른 강성의 폴리 아크릴 아마이드 젤 (PAAS)에 기초 invadopodia 및 견인력 세이를 이용하여 시험 관내에서 상관 될 수있다. 두 분석 사이에 변동이 존재하지만, 여기에 제시된 프로토콜 번째 PAAS를 만들기위한 방법을 제공한다AT는 두 분석에서 쉽게 사용될 사용자의 특정 생물학적 및 기술적 요구에 적응할 수있다.

invadopodia 분석에서, invadopodia은 전형적 전지 본체 내의 구조에 점 모양 cortactin 액틴과 같은 마커 (도 1)의 colocalization을함으로써 식별된다. 모두 적극적으로 저하 및 비 분해 invadopodia는 카운트 될 수 있으며, 이러한 구조는 FITC 표지 된 피브로넥틴 (도 1)에 형광 신호가없는, 검은 부분으로 colocalized 여부에 의해 구별된다. Invadopodia 수동 계산하고, 셀당 ECM 분해 수동 셀 내에 ?...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

종양 미세 환경에서 기계 강성이 invadopodia 활동을 증가시키고 수축력을 액토 마이 오신으로 악성 동작을 운전에 중요한 역할을한다. 사용하는 폴리 아크릴 아미드 겔 (PAAS)는, invadopodia 및 견인력 분석은 강성 매트릭스에 응답하여 암세포의 침윤 및 수축 특성을 연구하기 위해 이용 될 수있다.

암 세포에 Invadopodia 및 견인 포스 시험 법에 대한 폴리 아크릴 아마이드 젤

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Research

JoVE Journal

Medicine

12.0K 조회수.  Vanderbilt University Medical Center.  종양 미세 환경에서 기계 강성이 invadopodia 활동을 증가시키고 수축력을 액토 마이 오신으로 악성 동작을 운전에 중요한 역할을한다. 사용하는 폴리 아크릴 아미드 겔 (PAAS)는, invadopodia 및 견인력 분석은 강성 매트릭스에 응답하여 암세포의 침윤 및 수축 특성을 연구하기 위해 이용 될 수있다. 

비디오: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Stereotactic Radiosurgery for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3 Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4 patient mouthpiece active breathing coordination,5 respiration-correlated computed tomography,6 or image-guided tracking of fiducials implanted within and around a moving tumor.7-9 The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10 Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8 Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET).8 Use of 18F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.

Gynecologic 암에 대한 Stereotactic Radiosurgery의

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

연구

의학

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

정렬 줄기 세포 Assays 및 흐름에 대한 기본 뇌 종양 조직의 처리

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

설치류 비뇨 부비동 간엽 및 인간 줄기 세포의 조직 재조합을 사용하여 인간의 전립선 상피의 형성

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

임상 및 전임상 연구 응용 프로그램에 대한 종양 세포 (CTC) 분석 실험을 순환 반자동의 적응

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

고체 시편에서 기본 난소 암 세포를 얻는 방법

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

암 세포에 Invadopodia 및 견인 포스 시험 법에 대한 폴리 아크릴 아마이드 젤

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