Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

참고 : 동물과 인간의 암 세포가 구입하거나 친절하게 우리에게 제공 되었기 때문에 없음 윤리 고려. 1. 만들기 콜라겐 플러그 (의사 차 종양) <ol><li> 콜라겐 분산을 준비합니다. 이전에 17 일 또는 구입을보고 나도 추출하여 멸균 할 수 있습니다 쥐의 꼬리 힘줄 (RTT)에서 콜라겐 입력합니다. (; 다섯 2 분 실행 고속 설정) 균일 한 혼합을위한 동결 건조 된 RTT 콜라겐 (0.02 N 아세트산에 3.25-3.50 ㎎ / ㎖) 블렌더를 사용하여 분산. </li><li> 수확 (트립신 EDTA, 보통) 및 혈구를 사용하여 가능한 세포를 카운트 트리 판 블루 배제을 사용합니다. 원하는 세포 밀도 (플러그 당 5 × 10 4 세포)로 조정합니다. </li><li> 무균 조건 하에서 모든 솔루션 (수산화 나트륨, 소 태아 혈청, DMEM 5 배,의 NaHCO3) 별도로 (표 1)을 준비하고 얼음에 냉장 보관하십시오. 주 : 각종 용액의 첨가 순서는 셀 또는 산성 삼투압 충격을 방지하는 것이 중요하다. </li><li> 가능한 한 빨리 최종 콜라겐 용액 (5 mL)에 셀 분산액 (1.25 × 10 6 세포)를 수행한다. 기포를 방지하고 신속 96- 웰 플레이트의 각 웰에 즉시 사용 용액 200 μL를 배포하면서 (최대 피펫 아래쪽으로) 잘 섞는다. 부드럽게 기포를 제거하는 세포 배양 후드의 작업 영역 표면의 멀티 웰 플레이트를 찍는 웰 내부에서 균일 용액을 전파. </li><li> 모든 우물을 작성 후 인큐베이터에 보관 (이 단계는 96 웰 플레이트 당 약 15 ~ 20 분 소요). </li><li> 하룻밤에 2 시간에서 37 ° C에서 접시를 품어. 콜라겐 겔화 (즉, fibrillogenesis)는 30 분 이내에 발생합니다. 하룻밤 배양을 수행하기 위해 문화 문화 매체 (100 μL / 웰)를 추가합니다. </li></ol> 섬유소 젤 2. 첫 번째 레이어 <ol><li> 피브리노겐 용액 준비. 주 : 피브린 겔 같은 일괄 적 재현성 있습니다 END_STRONG_1 사용해야TS와 같은 피브린 겔 형성은 상업 동결 건조 피브리노겐의 다른 배치에 따라 다를 수 있습니다. <ol><li> 항상 새로 제조 피브리노겐 용액을 사용한다. 수화물 결정 형성을 방지하기 위해 유리 병을 열기 전에 실온으로 동결 건조 된 피브리노겐을 가지고. </li><li> 점진적으로 사전 예열 (37 ° C) 칼슘과 행크의 균형 소금 솔루션 (HBSS가) / 3 ㎎ / ㎖의 작업 농도의 Mg 2+ (최소 최종 부피의 15 % 초과를 준비 고려할 필요에서 피브리노겐을 용해 : 예를 들면, 5 ml의 솔루션에 대한 5.75 용액에 17.25 mg)을 얻었다. <ol><li> 피브리노겐 조각을 용해하기 위해 처음에 미리 예열 HBSS의 적하를 추가합니다. 비커에 주걱으로 더 큰 조각을 분해. 시간과 혼합 용이하게하기 위해 시간을 비커를 교반. 절차를 수행하는 동안 교반기 플레이트를 사용하지 마십시오. 위아래 정지를 피펫 팅하여 나머지 분말을 녹여. </li></ol></li><li> steri 동안 미지근한 피브리노겐 용액 유지0.22 ㎛의 필터에 통과시켜, 용액을 LIZING. 주 : HBSS 충분히 따뜻하지 않거나 피브리노겐이 완전히 용해되지 않은 경우, 솔루션은 필터를 막히게 할 수있다. 해당되는 경우, 피브리노겐 농도를 감소, 따라서 섬유소 응고 강성 수있는, 한 번 또는 두 번 필터를 교체합니다. </li><li> 다른 방법으로서, 최종 부피를 조절하면서 즉시 사용 피브리노겐 용액에 관심있는 세포 (예, 내피 세포)에 일시. </li></ol></li><li> 트롬빈 솔루션을 준비. <ol><li> DDH 2 O (50 NIH 단위 / ㎖)의 스톡 용액을 제조 한 후 0.22 ㎛의 필터를 사용하여 멸균. </li><li> 피브리노겐 / 트롬빈 비율 ≥1 사용 0.0075 (V / V)이 피브린 겔을 생성하기 위해서이다. </li></ol></li><li> 섬유소 젤을 생성. <ol><li> 멸균 피브리노겐을 유지하고 모든 다음 단계 동안 얼음에 재고 솔루션을 트롬빈. 섬유소 젤은 24 웰 플레이트에 형성 할 수 있습니다. </li><li> 즉시 오버레이 일각각의 E면 웰과 기포의 형성을 피하면서 피브리노겐 용액 (200 μL / 웰). 한 번에 6 우물을 처리합니다. </li><li> 피브리노겐 용액이 완전히 웰의 표면을 커버하면, 45 ° 각도로 플레이트를 기울 웰의 중심에 트롬빈 놓아 제 1 웰에 트롬빈 용액 1.5 μL를 추가 한 다음 부드럽게 대해 가로 판 소용돌이 1-2 초. <ol><li> 층류 후드 (5-10 분) 겔화 / 응고 과정이 완료 될 때까지 아래 안정 위치에서 상기 플레이트를 남겨 (NB : 중합 공정은 인큐베이터에 플레이트를 반송하여, 예를 들어 방해 안된다). </li></ol></li><li> 처음 여섯 우물이 중합 한 후 모든 우물이 처리 될 때까지, 다음 6 우물에 동일한 순서 (즉, 3 이전 단계)를 반복합니다. </li></ol></li></ol> 섬유소 젤과 샌드위치 콜라겐 플러그의 3 번째 레이어 <ol><li> 선택권A : (즉시 콜라겐 플러그를 사용). <ol><li> 섬유소 젤의 제 1 층 섬세 접시를 기울여 모든 우물에서 중합 것을 확인합니다. 콜라겐 플러그의 전송을 쉽게하기 위해 (섬유소 젤을 포함) 24 웰 플레이트 옆에 콜라겐 겔 플러그 측을 포함하는 96 웰 플레이트를 놓습니다. </li><li> 콜라겐 플러그를 함유하는 플레이트의 각 웰에 HBSS 한 방울을 추가한다. </li><li> 얇은 (핸들로 사용) 주사기에 장착 된 바늘 또는 (동영상 참조) 마이크로 스푼을 이용하여 우물에서 각 콜라겐 플러그를 제거합니다. 콜라겐 플러그 웰 웰에 중심을 그 불임성이 잘 유지되고 있는지 확인하면서, 하나 또는 두 개의 마이크로 숟가락을 사용하여 상기 제 피브린 겔 층에 각각 콜라겐 플러그를 옮긴다. </li><li> 피브리노겐 용액의 두번째 층이 미리 형성된 피브린 겔 오버레이 (300 μL / 웰) 및 2.3에 기재된 바와 같이 최소한 1 유지 트롬빈 소개 :. 한번에 0.0075 비율 여섯 웰 시퀀스 &lt;/ 리&gt; </ol></li><li> (성장 인자 - 감소 지하 막 (GFRBM)의 얇은 층으로 콜라겐 플러그 코팅) 옵션 B. <ol><li> 쿨 모든 준비 솔루션 및 악기 사전 및 GFRBM의 냉동 분취 이후 처리 과정 (예를 들면, 피펫, 조언, 테스트 튜브) 4 ° C 또는 얼음에 그들을 유지는 (제조업체의 지침에 따라) 해동시 과도한 가열 속도에 매우 민감하다 . </li><li> 플레이트 웰로부터 제거 후, ​​순수 GRFBM 용액 100 ㎕를 함유하는 얼음에 1.5 mL의 원심 분리 튜브에서 2 분 동안 각각 콜라겐 플러그 소크. </li><li> 전술 한 바와 같이 그것을 보장하는 것은 물론 중심 동안 첫 번째 섬유소 층에 각각 코팅 플러그를 전송합니다. 5 분의 GRFBM이 겔을 형성 할 수 있도록 37 ℃에서 플러그 - 함유 플레이트를 인큐베이션. 단계 3.1.4에서와 같이 두 번째 섬유소 층을 추가합니다. </li></ol></li></ol> 4. 세포 배양 중간 조건 <ol><li> (배지와 잘 4 각 채우기00 μL). 배양액 및 보조제는 세포주 및 실험 조건에 따라 선택 될 것이다. </li><li> 100 칼리 크레인 저해 단위 (KIU) / ㎖의 최종 농도로 배양 배지에 아프로 티닌, antifibrinolytic 제를 추가한다. 주 : 시험 된 세포주에 이용되는 조건 하에서 세포 배양 인큐베이터에서 플레이트를 저장. </li><li> 매일 신선한 매체와 문화를 보충하거나 실험 일정에 따라, 그리고 아프로 티닌을 추가합니다. 새로운 배지를 추가하기 전에 약간의 (a 30 ~ 35 ° 각도로) 판을 기울 조심스럽게 일정한 관찰에서 조정 배지를 흡입하는 동안 잘의 측면에 대해 피펫을 경사. </li></ol>

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S., Degen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+and+Tumor+Cell+Metastasis.">Fibrinogen and Tumor Cell Metastasis.</a> Haemostasis. 31, 11-15 (2001).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Stroma,+Tumor+Blood+Vessels,+and+Antiangiogenesis+Therapy.">Tumor Stroma, Tumor Blood Vessels, and Antiangiogenesis Therapy.</a> Cancer J. 21 (4), 237-243 (2015).</li><li>Luoto, K. R., Kumareswaran, R., Bristow, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Hypoxia+as+a+Driving+Force+in+Genetic+Instability.">Tumor Hypoxia as a Driving Force in Genetic Instability.</a> Genome Integr. 4 (1), 5 (2013).</li><li>Das, V., Bruzzese, F., Kone&#269;n&#253;, P., Iannelli, F., Budillon, A., Hajd&#250;ch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+In+Vitro+Tumor+Models+for+Drug+Screening.">Pathophysiologically Relevant In Vitro Tumor Models for Drug Screening.</a> Drug Discov Today. 20 (7), 848-855 (2015).</li><li>Longati, P., 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=3D+Pancreatic+Carcinoma+Spheroids+Induce+a+Matrix-rich,+Chemoresistant+Phenotype+Offering+a+Better+Model+for+Drug+Testing.">3D Pancreatic Carcinoma Spheroids Induce a Matrix-rich, Chemoresistant Phenotype Offering a Better Model for Drug Testing.</a> BMC Cancer. 13 (95), (2013).</li><li>Tan, P. H., Chia, S. S., Toh, S. L., Goh, J. C., Nathanm, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Spatial+Configuration+of+Tumour+Cells+Confers+Resistance+to+Chemotherapy+Independent+of+Drug+Delivery.">Three-dimensional Spatial Configuration of Tumour Cells Confers Resistance to Chemotherapy Independent of Drug Delivery.</a> J Tissue Eng Regen Med. , (2013).</li><li>Koutsilieris, M., Reyes-Moreno, C., Choki, I., Sourla, A., Doillon, C., Pavlidis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+Cytotoxicity+of+Human+MCF-7+and+MDA-MB+231+Breast+Cancer+Cells+is+Altered+by+Osteoblast-Derived+Growth+Factors.">Chemotherapy Cytotoxicity of Human MCF-7 and MDA-MB 231 Breast Cancer Cells is Altered by Osteoblast-Derived Growth Factors.</a> Mol Med. 5 (2), 86-97 (1999).</li><li>Lang, 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=Biphasic+Response+of+Cell+Invasion+to+Matrix+Stiffness+in+Three-Dimensional+Biopolymer+Networks.">Biphasic Response of Cell Invasion to Matrix Stiffness in Three-Dimensional Biopolymer Networks.</a> Acta Biomater. 13, 61-67 (2015).</li></ol>

The authors have no disclosure.

중요한 기술적 각주, 갭 중앙과 주변 겔 사이의 계면에 존재하지 않는 것이 중요하다. 그렇지 않으면 / 마이그레이션 피브린 겔 침투하는 전지의 용량을 줄일 수있다. 트롬빈 적절히 희석되지 않은 경우 콜라겐과 피브린 겔 사이의 공간은 배양의 처음 24 시간 동안 형성 할 수도있다. 콜라겐 겔을 초래할 수 시험 세포주 모두시켜 겔 사이에 형성하기 위해 상대적으로 큰 공간을 일으키는 동안 배양 ...

암 세포 침공 / 마이그레이션 및 후속 전이 설립의 기본 및 생물 의학 특성을 조사하는 강렬한 연구 1, 2의 주제이다. 전이 암의 최종 단계이며, 임상 관리하기 어려운 남아있다. 세포 및 분자 수준에서의 전이에 대한 이해는보다 효율적인 치료 (3)의 개발을 가능하게한다. 전이성 세포의 여러 특성은 자신의 stemness 및 마이그레이션 내에서 기본 종양 5 침공 전이 상태 (예를 들면, 상피 - 중간 엽 전이를) 획득 가능성을 포함하여 시험 관내 4 탐험 할 수 있습니다. 그것은 거의 혈액 / 림프 순환의 기여를 배제하기 때문에, 내습 / 전이 과정의 시험 관내 평가는 도전하고있다. 콜라겐 젤에 종양 조각을 삽입의 Organotypic 문화는 기존에 가지고교활한 암의 공격성을 모니터링하는 데 사용되었다. 종양의 복잡도가 보존되어 있지만 (예를 들면, 비 - 암성 세포의 존재), 종양 절편 샘플링 변동 제한된 확산 매체에 노출 및 기질 세포 (6)의 과도한 성장한다. 대안적인 방법은 3 차원 셀 환경을 모방 세포 외 기질 (ECM)의 구성 요소 내에서의 암세포 성장에있다. 콜라겐 겔 및 / 또는 기저막 유래 매트릭스 유방암 세포주의 증식 차원 세포 배양의 가장 특징으로하는 실시 예 사이이다. 특정 3 차원 세포 배양 환경을 사용하여 표준 조건 하에서 성장 유방암 세포에 대해 관찰 된 무질서 조립체 유방 꽈리 및 관상 구조 7-10의 자연 형성에 역전 될 수있다. 또한, 선암종 암세포 유래의 다세포 종양 타원체의 형성은 (서로 다른 기술을 이용하여 군집예를 들면, 현재 가장 일반적으로 사용되는 3 차원 세포 배양 분석법 11-13을 구성하는) 매립 한천, 타원체, 플로팅 방울 매달려. 그러나 이러한 분석은 구 상체를 형성 할 수있는 암 세포주의 제한된 세트에 의해 이러한 조건에서 세포를 연구 할 수 짧은 기간에 의해 제한된다. 이러한 시각화 방법에서, 우리는 본 명세서에서 관심있는 암세포 대안 기저막 유래 매트릭스로 피복 될 수있는 의사 원발 종양의 체외 형성을 허용하기 위해 콜라겐 겔에 포함 된 정교한 3D 세포 배양 분석을 소개한다. 형성되면, 가상 기본 종양 후 암세포 두 매트릭스 구획과의 계면을 통과 할 수있는 무 세포 기질 (본 경우 피브린 겔)에 끼워진다 (도 1 참조). 흥미롭게 공격적인 암세포와 함께 의사 원발 종양 유래 이차 종양 같은 구조가 나타나지섬유소 젤. 이러한 3 차원 배양 시스템은 예를 들어, 항암제, 유전자 발현 및 세포 - 세포 및 / 또는 세포 ECM 상호 14-16 조사하는 필요한 유연성을 제공한다. <img alt="그림 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 그림 1 :.. 방법의 개요 암 연구를위한 모델로 3D 세포 배양 시스템을 생성하는 방법의 도식 요약 <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table><tbody><tr><td>Freeze-dried collagen</td><td>Sigma-Aldrich</td><td>C7661</td><td>from rat tail tendon (soluble dispersion) or home-made (see Rajan &lt;i&gt;et al&lt;/i&gt;., ref.#14)</td></tr><tr><td>Fibrinogen (freeze-dried)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F8630</td><td>Type I-S, 65 - 85% protein with &amp;ge; 75% of protein is clottable</td></tr><tr><td>Thrombin</td><td>EMD Chemicals Inc.</td><td>605157</td><td>&amp;nbsp;Gibbstown, NJ; NIH units/mg dry weight&amp;nbsp;</td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>Corning</td><td>356234</td><td>Previously from BD Biosciences</td></tr><tr><td>Aprotinin</td><td>Sigma-Aldrich</td><td>A6279&amp;nbsp;</td><td>&amp;nbsp;solution at 5 - 10T IU/ml (Trypsin Inhibitor Unit)&amp;nbsp;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Micro-spoons</td><td>Fisher Scientific</td><td>2140115</td><td>Fisherbrand Handi-Hold Microspatula</td></tr><tr><td>96 well plate, round base</td><td>Sarstedt</td><td>3925500</td><td></td></tr><tr><td>24 well plate</td><td>Sarstedt</td><td>3922</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s Medium</td><td>Sigma Chemical, Co.</td><td>D5546</td><td>DMEM</td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>CAA15-701</td><td>FBS, Canadian origin.</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Chemical, Co.</td><td>T4049</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution&amp;nbsp;</td><td>Sigma Chemical, Co.</td><td>H8264</td><td>HBSS</td></tr></tbody></table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

단일 층에서 포유 동물 세포 배양 널리 다양한 생리 학적 및 분자 과정을 연구하는 데 사용됩니다. 그러나 성장 세포를 연구하는이 방법은 종종 원치 않는 아티팩트를 생성합니다. 따라서, 주로 세포 외 기질 성분을 사용하여 3 차원 (3D) 환경에서 세포 배양이 때문에 생체 조직 또는 기관에서 고유로 그 유사성 흥미로운 대안으로 나타났다. 우리는 피브린 겔 이루어지는 의사 차 macrospherical 종양 및 (ⅱ) 주연 무 세포 격실 콜라겐 겔 연기에 포함 된 암 세포를 함유하는 중앙 구획 개의 구획, 즉 (i)를 이용하여 3 차원 세포 배양 시스템을 개발 즉, 센터에서 사용되는 다른 세포 외 매트릭스 성분이있는 암세포 마이그레이션 (내습 전면) 및 / 또는 보조 위성을 나타내는 종양 microspherical 종양을 형성 할 수있다. 주변 구획 위성 종양의 형성은이 현저 차원 배양 시스템의 차별화 네이티브 종양 세포의 공지 공격성 ​​또는 전이성 기원 상관. 이 세포 배양 방법은 암 세포의 침윤과 운동성, 세포 외 기질의 상호 작용 및 항암 약물의 특성을 평가하는 방법으로 평가하는 것으로 간주 될 수있다.

앞서 언급 한 바와 같이,이 3 차원 세포 배양 검정의 흥미로운 특징은 암세포에만 인접 피브린 겔 콜라겐 플러그 마이그레이션뿐만 아니라, 이차 종양 (예, 위성 종양 형 구조)를 확립 할 수 있다는 것이다. 이는 직접 특히 긴 작동 거리 응축기 (도 2)과, 겔의 두께를 통해 저 및 고 배율에서의 역 위상차 현미경으로 관찰 할 수있다. 다른 실험 셋업 (15)?...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

암 세포 침윤 및 위성 종양 형성을 탐색하는 문화 분석 세 가지 차원

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

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10.4K 조회수.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

비디오: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

연구

의학

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

침공을 공부 하 고 방광 암에서 치료제를 평가 하기 위한 3 차원 셀 문화 시스템

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

암 연구학

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D 배양에서 암 세포 침략 및 T 세포 세포 독성 을 감시

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

세 가지 차원 문화 : 유방 세포의 악성 변형 대 일반 선포 건축을 연구하는 도구

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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

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

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

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

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

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

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

종양 기질 상호 작용에 대한 세 가지 차원의 공동 문화 모델

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

상피암 세포의 스캐폴드가 없는 3D 배양에서 이종다세포 기질 세포(Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion)

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

암 세포 회전 타원체 분석은 3D 설정에 침략을 평가하기 위해

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment.

3D 배양 기저막 분석: 세포 침입을 연구하기 위해 3D 기저막 단백질 매트릭스에서 암세포 배양

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...