We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

설명하는 모든 연구 프로토콜은 시카고 임상 시험 심사위원회의 대학 (IRB)에 의해 검토되었다. 동의서는 수술 전에 각 환자로부터 얻어진 것으로,이 연구는 시카고 IRB의 대학에 의해 승인되었다. 보호를위한 인체 조직을 처리하고 세포를 오염의 위험을 줄일 때 생물 안전 캐비닛 2 형 장갑을 사용해야합니다. 차 변환되지 않은 기질 세포의 1. 분리 및 문화 <ol><li> 인체 조직 수집 및 준비. <ol><li> 복부 수술 중 제거 된 인간의 대망, 2cm 3의 표본을 확보, 즉시 RT 인산 완충 식염수 (PBS) (그림 2A)에서 조직 몰입. X 2cm 대망 3cm의 조각은 일반적으로 1,000,000 주 인간 중피 세포 (HPMC) 및 20 차 인간 섬유 아세포 또는 정상 대망 섬유 아세포 (NOF)를 산출한다. </li><li> 조직이 3 0.5 XG에 침지 PBS를 스핀 다운분 가능한 한 빨리 후 수집 (&lt;2 PBS의 시간)과 50 ML 원뿔 튜브에 신선한 20 ml의 PBS로 조직을 전송합니다. 15cm 직경, 무균 배양 접시 (그림 2B)에서 메스로 닦지에 의해 5mm 3 조각으로 조직을 잘라. </li></ol></li><li> 일차 인간 중피 세포 분리 8 <ol><li> HPMC를 분리하려면 50 ML 원뿔 튜브에 다진 조직을 전송하고 고체 조각이 상단 (그림 2C &amp; D)에 떠있는 1 분을 기다립니다. HPMC와 적혈구 (RBC)의 하단에있는 액체에 존재합니다. 튜브의 바닥에서 그리고 새로운 원추형 튜브로 액체를 제거하기 위해 피펫을 사용한다. 3 분 0.5 XG에서 액체를 스핀 다운 및 HPMC / RBC 펠렛에서 뜨는을 대기음. <ol><li> , 다진 조직에 20 ml의 PBS를 추가 조직, 스핀 다운, 상승 피펫 및 HPMC / RBC 튜브에 바닥에서 액체를 제거 할 수 있도록하고 제거 : 과정을 세 번 반복상층 액. 모든 스핀 완료 및 상청액 흡입 후, 펠릿 HPMC 및 RBC를 포함 할 것이다. </li></ol></li><li> 플레이트 10 % 소 태아 혈청 [FBS, 1 % MEM 비타민 [93 ㎎ / ℓ], 1 % MEM 비 필수 아미노산 [81.4 전체 성장 배지 15 ㎖ (DMEM에서 75cm 2 플라스크에 펠릿의 모든 셀 ㎎ / ℓ], 1 % 페니실린 스트렙토 마이신 [펜 연쇄상, 100 U / ㎖ 페니실린 및 100 μg의 / ㎖ 스트렙토 마이신). </li><li> 추가 HPMCs을 분리 보조 PBS 세척을 수행합니다. <ol><li> 조직이 정상에 떠위한 보조 PBS 세척를 들어, 200 rpm으로, PBS의 20 ㎖에서 10 분 동안 37 ° C에서 남아있는 고체 조직을 흔들어 후 1 분을 기다립니다. 3 분 0.5 XG에 원심 분리기, 새로운 튜브로 튜브의 바닥에서 액체를 제거하고 뜨는을 대기음. </li><li> 전체 성장 매체의 15 ㎖에 별도의 75cm 2 플라스크에 씻어 보조 PBS에서 모든 HPMC / RBC 플레이트. </li></ol></li><li> 어떤 연구를 분리하려면부피 PBS 용액 : 1 0.25 % 트립신 25 밀리미터 EDTA : HPMC를 emaining, 1 20ml에 10 분간, 200 rpm에서 37 ° C 정도를 다진 조직을 흔들. 조직, 상승 관의 하단에있는 액체를 수집 새로운 50 ML 튜브로, 0.5 GX 3 분에서 스핀 다운을 허용합니다. <ol><li> 뜨는을 대기음 및 전체 성장 매체의 15 ㎖에 별도의 75cm 2 플라스크에 모든 HPMC / RBC를 접시. </li></ol></li><li> 배양 습한 환경에서 37 ° C에서 5 % CO 2에서 도금 셀. 소비 매체를 제거하지 않고 3 일 및 5 일에 완전 성장 배지 15 ㎖에 첨가함으로써 공급 도금 셀. HPMC는 분할 전에 5-7일 배양 할 수있다. <ol><li> 원래의 표현형의 탈분화 및 수정을 최소화하기 위해 모든 실험에서 (통로 2까지) 낮은 통로 HPMC를 사용합니다. 위상차 capabilitie 플라스틱 미분 간섭 대비 기능 또는 4-20x 목적으로 20 배 목적으로 바람직 넓은 필드 현미경을 사용하여의 세포의 모든 이미지를 복용 (현미경의 위상 콘트라스트 필터), 3,3&#39;- 디아 미노 (DAB) 얼룩을 면역 조직 화학 분석 밝은 분야에서 10 배 목표 (). </li></ol></li><li> HPMC가 입방 것을 확인하고 면역 조직 화학 (8) (그림 3A, C, E)를 수행하여 cytokeratin 8 멘틴을 표현한다. </li></ol></li><li> NOF 분리 (8) <ol><li> 1 : 100 배 펜 연쇄상 구균의 1 : 1 혼합물 (1)를 결합하여 콜라겐 유형 III 솔루션 (10 배)의 주식 10 ㎖를 준비 콜라게나 제 3 형의 1,500 유닛 활동 / ㎖를 : PBS에서 히알루의 714 유닛 활동 / ㎖. </li><li> 1 % MEM 비타민 [93 ㎎ / ℓ]와 무 혈청 배지에서 희석 10 배 콜라겐 유형 III 용액 10 ㎖ (DMEM에 6 시간 동안 200 rpm에서 HPMC 분리 후 남아있는 다진 대망의 조직, 37 ° C를 흔들어, 1 % MEM 비 필수 아미노산 [81.4 ㎎ / ℓ], 1 % 페니실린 스트렙토 마이신 [펜 연쇄상, 100 U / ㎖ 페니실린 및 100 μg의 / ㎖ 스트렙토 마이신]). 소화 조직은 불투명 한 모양과 일부 섬유 파편 (그림 2E)을 가질 수있다. </li><li> NOF 함유 용액을 원심 분리하여 세포 실온에서 3 분 0.5 XG에 정지에, 뜨는을 대기음 및 전체 성장 매체의 13 ㎖에 75cm 2 플라스크에 펠렛을 접시. </li><li> 이전 용지를 제거하고 24 시간 후 신선한 전체 성장 매체의 15 ㎖로 대체합니다. NOF는 분할 전의 1-3일 배양 할 수있다. 세포가 포화 상태에 도달했을 때 NOF의주의 확산은 중단됩니다. </li><li> 기본 인간 섬유 아세포는 평면, 신장 세포임을 확인하고 멘틴을 표현하지만, 면역 조직 화학 (8)을 수행하여 8 cytokeratin하지 (그림 3B, D, F). 탈분화을 최소화하기 위해 (이상적 통로 (3) 전) 실험 낮은 통로 NOF를 사용하여 원래 표현형의 수정. 세포의 모든 단계 이미지를 복용 플라스틱 DIC 기능이있는 20 배 목적으로 넓은 필드 현미경을 사용하여,밝은 분야에서 10 배의 목표는 면역 조직 화학 DAB 염색을 분석합니다. </li></ol></li></ol> 2.의 Organotypic 문화 (8) 도금 <ol><li> 더 이상으로 5 분 이상 3 ㎖의 0.25 % 트립신 / EDTA 25 mM의 뒤에 PBS 10 mL로 세척하여 75cm 배양 플라스크에서 분리 NOF. 완전 성장 배지의 적어도 3 배 부피의 트립신을 중화. </li><li> 50 ML 원뿔 튜브에 트립신 세포를 전송하고 0.5 GX 3 분에서 스핀 다운. 상층 액을 제거하고 전체 성장 미디어 5 ㎖에 다시 세포를 가지고. 문화 플라스크에서 회수 된 세포를 계산하는 셀 카운터를 사용합니다. </li><li> 96 웰, 분명 바닥, 검은 접시에 100 μL 당 2,000-4,000 NOF를 판에 전체 성장 매체의 적절한 볼륨에서 세포를 희석. 도금하기 전에 세포 혼합물에 쥐 꼬리 콜라겐 I는 0.5 μg의 / 100 μl를 추가합니다. 하자 세포는 적어도 4 시간 동안 습한 환경에서 5 % CO 2에서 37 ° C에 앉아서, 또는 세포까지플레이트 표면에 부착합니다. </li><li> 단계 2.1 및 2.2에 설명 된 것과 동일한 방법을 사용하여 배양 플라스크에서 HPMC를 놓습니다. 완전 성장 배지의 적당한 양의 세포를 이미 96- 웰 플레이트에서 콜라겐 I 도금 NOF의 상부에 50 μL 당 10,000-20,000 HPMC를 플레이트에 희석. 세포 실험을 시작하기 전에 적어도 18 시간 동안 습한 환경에서 5 % CO 2에서 37 ° C에서 앉게. </li><li> 완전 성장 배지에서 HeyA8 난소 암 세포를 배양 발현 녹색 형광 단백질 (GFP). HeyA8 난소 암 세포가 형광 요각류 cGFP (pCDH-CMV-MCS-EF1)를 발현하는 렌티 바이러스 벡터의 식으로 표시되어 있습니다. HeyA8 난소 암 세포는 사용 일 (대수 증식기)에 80-90% 합류해야한다. 배양 접시에서 세포를 분리 및 일차 전지에 대한 단계 2.1-2.2에 설명 된 것과 동일한 방법을 사용하여 계산합니다. </li><li> 난소 암 세포는 37 ℃에서 15-20 분 동안 완전 성장 배지로 복구 할뚜껑을 원뿔 튜브 느슨하게 봉인. </li></ol> 3. 접착 분석 (8) <ol><li> 회복 후, 0.5 XG에서 3 분 동안 회전시켜 난소 암 세포 펠렛 후 상청액을 제거하고 50,000 세포 / 100 ㎕의 농도를 달성하기 위해 혈청이없는 배지의 적당한 부피 세포를 희석. </li><li> 소비 매체를 분리 HPMC / NOF의 Organotypic 함께 배양 96 웰 플레이트를 전환하고, 각 웰에 무 혈청 배지에서 암 세포 현탁액 100 ㎕를 추가한다. 4 시간 30 분 동안 습한 환경에서 5 % CO 2에서 37 ℃에서 플레이트를 배양한다. </li><li> 미디어 및 비 - 부착 세포를 제거하기 위해 96 웰 플레이트를 전환. 조심스럽게 부드럽게 잘 각각에 PBS의 100 μl를 피펫, 다시 판을 반전 낮은 전력에서 멀티 채널 피펫을 사용합니다. PBS를 한 번 더 씻어 반복합니다. </li><li> PBS를 제거하고 각 웰에 100 ㎕의 4 % 파라 포름 알데히드를 추가하는 반전. 판은 20 마일 동안 앉아 허용N 세포를 해결하려면. 20 분 후, 플레이트를 반전 100 μl의 PBS를 추가합니다. </li><li> 웰의 총 형광보고 될 수 있거나 또는 세포의 수를 정량 할 수 있습니다. 정량, 1 백만 세포 / ml의 농도로 HeyA8 세포를 이용한 복제에서 제 1 플레이트 표준 곡선. <ol><li> 아래 100 만 셀을 회전 미디어를 제거하고 20 분 동안 1 ml의 4 % 파라 포름 알데히드에 앉아 할 수 있도록하여 표준 곡선을 확인합니다. </li><li> 스핀 세포는 다시 PBS (재검 세포가 100 만 / ml의 농도를 확인하기 위해) ㎖로 1에서 가져온다. 플레이트 50,000, 40,000, 30,000, 20,000, 10,000, 5,000, 1,000, 잘 중복의 각에 0 세포 및 각 웰에 50 μL의 최종 총 볼륨 PBS를 추가합니다. </li></ol></li><li> 아래는 높은 우물 (표준 곡선에 50,000) 확장, 형광 플레이트 리더 (여기 = 488 nm의, 방출 = 528 ㎚)의 문화 판을 읽어 보시기 바랍니다. organotyp 부착 얼마나 난소 암 세포를 계산하는 표준 곡선을 사용하여각 웰 (도 4A-C)에서 IC 배양. </li></ol> 4. 대량 살상 무기 확산 분석 (10) <ol><li> 난소 암 세포가 회복 후 1 % FBS와 (DMEM에게 1 % FBS 배지의 적당한 부피로 세포를 희석 한 다음 0.5 XG에서 3 분 동안 회전시켜 세포 펠렛과, 1 % MEM을 (단계 2.5 이상 2.6 참조) 비타민 [93 ㎎ / ℓ], 1 % MEM 비 필수 아미노산 [81.4 ㎎ / ℓ], 1 % 페니실린 스트렙토 마이신 [펜 연쇄상 구균은 100 U / ㎖ 페니실린 및 100 μg의 / ㎖ 스트렙토 마이신])를 4,000 세포의 농도를 달성 / 150 μL. </li><li> 각 웰에 FBS 매체를 소비 매체를 제거하고, 1 %에서 암 세포 현탁액 150 μl를 추가 HPMC / NOF의 Organotypic 문화로 96 웰 플레이트를 전환. 72 시간 동안 가습 환경에서 5 % CO 2에서 37 ℃에서 플레이트를 배양한다. </li><li> 아래는 상대적 확산을 평가하기 위해 한 번 형광 플레이트 리더 일 (여자 = 488 nm의, 방출 = 528 ㎚)의 문화 판을 읽고세포의. 48 시간 (그림 5A-C)에서 미디어를 변경, 96 시간 동안 분석을 계속합니다. (접시 바람직하다 반전) 미디어를 변경할 때 문화를 방해하지 않도록주의하십시오. </li></ol> 5. 침략 분석 (8) <ol><li> 3D 문화를 도금하기 전에, 24 웰 배양 플레이트 삽입 (8 μm의 기공 크기)에 200 μL PBS에 7.5 μg의 쥐 꼬리 콜라겐 I를 추가합니다. 판을 품어 O는 / N은, 조심스럽게 즉시 콜라겐 코팅 인서트 (위의 2 단계)에 HPMC / NOF의의 Organotypic 문화를 도금하기 전에 피펫으로 PBS를 제거 할 수 있습니다. </li><li> 단계 3.1에서와 난소 암 세포를 준비한다. 삽입에의 Organotypic 문화에 난소 암 세포를 플레이트에 조심스럽게 삽입의 상단에서 초과 용지를 제거하기 위해 피펫을 사용합니다. 각 웰에 무 혈청 배지에서 난소 암 세포 현탁액 100 ㎕를 추가한다. </li><li> 암 세포를 유도하는 웰 인서트 아래로 완전 성장 배지 750 μl를 추가의 Organotypic 문화에 vasion. 24 시간 동안 가습 환경에서 5 % CO 2에서 37 ℃에서 플레이트를 배양한다. 주 : 문화의 Organotypic 삽입물을 건너 인서트의 바닥면에 남아 침입 난소 암 세포. </li><li> 빈 우물에 삽입물을 이동 한 후, 24 시간 후, 집게와 인서트를 잡고 간단히 PBS의 비이커에 씻어. 1 분의 총 면봉의 양측 각 인서트의 상부 스크럽. 신속하게 잘 각각 750 μL 4 % 파라 포름 알데히드를 포함하는 플레이트에 삽입을 배치합니다. 각 삽입 750 μL PBS로 가득 우물에 삽입을 전송하기 전에 10 분 동안 파라 포름 알데히드에 앉아하도록 허용합니다. </li><li> 2.5 배의 배율로 현미경 침략 세포 (인서트의 바닥에 접착 고정)를 조회. 전체 웰이 시야 내에있는 경우, 회피, 10 배의 배율을 조정하고 5 사진, 물론 각 사분면에서 하나의 중앙 부근에 1을가장자리에 너무 가까이있는 이미지를 복용. 물론 각각의 동일한 패턴을 따릅니다. </li><li> 이들 프로토콜에 따라 각 웰 (도 6A-C)에서 필드 당 침략 세포의 평균 수를 얻기 위해 각각의 이미지 셀의 수를 계산하기 위해 제조자의 프로그램을 사용한다. </li></ol>

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K., 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

복막 미세 환경의의 Organotypic 모델은 난소 암의 보급에 미세 환경의 세포와 타고난 구성 요소를 모두의 개인 및 집단 기능 (들)을 평가하기 위해 설립되었다. 난소 암 세포 부착, 증식, 그리고 침략을 조사하기 위해 3 차원의 Organotypic 문화를 도금 및 사용자 정의에 대한 특정 프로토콜이 제공됩니다. 일차 인간 장간막 중피 세포 및 섬유 아세포는 환자로부터 분리 된 형태 및 정상 변이 환자를 보존 ...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table><tbody><tr><td>&lt;strong&gt;1. Isolation and culture of primary cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>Single-edged razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>Glass flask</td><td>?</td><td>?</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000044_3616914956</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker&amp;nbsp;</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td><td></td></tr><tr><td>Trypsin EDTA, 1x (0.25%)</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical</td><td>LS002592</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Plating 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>Countess</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Invitrogen</td><td>C10313</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Collagen Type I (Rat Tail)</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>96 well plate, clear bottom, black</td><td>BD Biosciences</td><td>353219</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Adhesion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multichannel pipet</td><td>Eppendorf</td><td>Xplorer 300</td><td></td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>Minimax</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;5. Invasion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Inserts (8um, 24-well)</td><td>BD Biosciences</td><td>353097</td><td></td></tr><tr><td>Cotton swabs</td><td>Q-tips</td><td>cotton swabs</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axiovert 200m</td><td></td></tr><tr><td>Cell Profiler</td><td>public domain</td><td></td><td></td></tr><tr><td>24 well plate</td><td>BD Biosciences</td><td>353047</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;6. Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Integrin &amp;alpha;V&amp;beta;3 Antibody, clone LM609</td><td>EMD Millipore</td><td>MAB1976</td><td></td></tr><tr><td>Beta 1&amp;nbsp;</td><td>Oncosynergy</td><td>OS2966</td><td></td></tr><tr><td>Alpha 5 [CD49e]</td><td>ID Pharmingen</td><td>555615</td><td></td></tr><tr><td>Beta 4 [CD104]</td><td>EMD Millipore</td><td>MAB 2058</td><td></td></tr></tbody></table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

배양의 Organotypic 제 콜라겐 타입 I과 차 인간 섬유 아세포를 혼합 한 다음 중피 세포의 5 배 번호 이것 문화를 오버레이하여 조립 하였다. 난소 암 세포 부착, 침입이나 확산을 연구하기 전에 첨가하고 배양은 적어도 18 시간 동안 인큐베이션 하였다. 각 분석은 다중 반복 하였다 (N = 5), 침윤 분석 (N = 3) (N = 3-5) 다른 환자 및 수많은 웰로부터 얻은 3D 배양 접착 각 조건에서 시험 하였다 (N = 5), 확산 . 4 ...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

인간의 복강의의 Organotypic 문화 난소 암 보급의 초기 단계 모델링

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

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JoVE Journal

Medicine

26.5K 조회수.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

비디오: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

원래 종양 개발의 쥐 실험 모델 및 복막 전이<em&gt;를 통해</em난소 암 세포에&gt; 동소 이식 접종

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

연구

암 연구학

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

의학

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

3 차원 Heterotypic<em&gt; 체외에서</em난소 암 개시 및 진행하는 동안 Stromal - 상피 조직의 상호 작용&gt; 모델링

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

난소암-오멘텀 상호작용을 위한 3D Ex Vivo 모델 구축

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

저자 스포트라이트: 암-지방 미세환경 상호작용 조사를 위한 고급 생체 외 모델 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

인간 Omentum을 사용한 난소암 복막 전이의 생체 외 모델

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

동적 3D 복막의 마이크로 디바이스의 모델링 난소 암 다세포 회전 타원체 동작

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

생체공학

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Source: Zink, K. E. et. al. <a href="https://www.jove.com/t/59490">Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,&#160; to detect small molecule exchange between the cells and tissues leading to tumor development.

이미징 질량 분석 분석을 위한 난소암 커뮤니케이션 모델링: 전이성 난소 종양 발달에서 소분자 화학 전달자를 식별하는 방법

Source: Zink, K. E. et. al. Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry. J. Vis. Exp. (2019)
This ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Assays & techniques

Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

Source: Peters, P. N. et al. <a href="https://www.jove.com/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the development of&#160; an in vitro &#160;three-dimensional model of the lining of the peritoneal cavity. This organotypic culture can help to investigate ovarian cancer cell adhesion, invasion, and proliferation.

복강내막의 난소암 전이 모델링: 복막강 내막의 체외 3D 모델을 확립하는 방법

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

인간의 복강의의 Organotypic 문화 난소 암 보급의 초기 단계 모델링

요약

더 많은 비디오 탐색

이 비디오의 챕터

원래 종양 개발의 쥐 실험 모델 및 복막 전이

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

3 차원 Heterotypic

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

인간 Omentum을 사용한 난소암 복막 전이의 생체 외 모델

인간 Omentum을 사용한 난소암 복막 전이의 생체 외 모델

인간 Omentum을 사용한 난소암 복막 전이의 생체 외 모델

동적 3D 복막의 마이크로 디바이스의 모델링 난소 암 다세포 회전 타원체 동작

이미징 질량 분석 분석을 위한 난소암 커뮤니케이션 모델링: 전이성 난소 종양 발달에서 소분자 화학 전달자를 식별하는 방법

복강내막의 난소암 전이 모델링: 복막강 내막의 체외 3D 모델을 확립하는 방법