MB는 MRC의 교제에서 보조금에 의해 지원됩니다. KP와 AHM은 웨 섹스 의학 연구 및 암 연구 UK / RCS (영국) (C28503/A10013)에서 보조금에 의해 지원된다. 우리는 사우 샘프 턴 조직 화학 연구 단위의 대학의 지원에 대한 감사합니다.

대장의 Explants에서 주요 섬유 아세포 문화 1. 설립 <ol><li> 직접 수술 수술실에서 정상적인 인간의 대장 점막 조직의 샘플을 확보, 7 ~ 10에서 중단 PBS의 ML 100 단위 / ML 페니실린, 100 ㎍ / ml의 스트렙토 마이신, 및 / ㎖ Fungizone 0.25 μg로 보충. </li><li> 실험실에서, 10-cm 조직 배양 접시의 중앙에 시료를 배치하고 PBS / 펜 연쇄상 / Fungizone로 3 회 세척 하였다. 최종 세척 후 흡인하지 마십시오. </li><li> 멸균 집게와 메스로 (약 2 mm) 작은 조각으로 조직을 잘라 12 - 웰 플레이트에 메스 블레이드와 그린 크로스의 중심에 각 조각을 배치합니다. 이 준수 할 수 있도록 조직에 부드럽게 메스 록. </li><li> 농경 750 μL의 시료는 일차 섬유 아세포 성장 배지 subme한다 (DMEM은 20 % 송아지 태아 혈청 (FCS), 100 U / ㎖ 페니실린, 100 ㎍ / ㎖의 스트렙토 마이신 및 292 ㎍ / ml의 L-글루타민)플로트를 사용하지 않고 조직을 RGE. 2 일마다 재 공급을 제외하고 향후 5 일 동안 플레이트를 방해하지 마십시오. </li><li> 향후 5 일 동안 섬유 아 세포는 천천히 조직의 가장자리에서 밖으로 성장하기 시작합니다. 대략 7 일째에, 이것은 더 볼 수 있습니다. 밖으로 성장 충분한 섬유 아 세포가 있는지 후 약 3 ~ 4 주와, 각 웰에 750 ㎕의 트립신을 추가합니다. 세포는 T-25 조직 배양 플라스크에, 수영장 및 전송 (5 ~ 10 분의 시간이 걸릴 수 있음) 분리 한 후. </li></ol> 2.의 Organotypic 준비 섬유 아세포 임신의 Organotypic 겔의 제조 : <ol><li> 10 % FCS 및 292 ㎍ / ml의 L-글루타민이 보충 된 DMEM에서의 Organotypic 당 5 × 105 세포를 포함하는 섬유 아세포의 세포 현탁액을 준비한다. </li><li> 다음과 같은 비율로, 얼음의 Organotypic 젤 메이크업 : <ul><li> 쥐 꼬리 콜라겐의 7 권 : 리겔의 혼합 (1:1) </li><li> 필터링 10 배 DMEM의 1 볼륨 </li><li> FCS의 1 볼륨 </li><li> 5 × 5 섬유 아세포를 함유하는 섬유 아세포의 세포 현탁액 1 부피 9 젤 (젤 당 1 ㎖)의 경우 10 ㎖를 준비하고 거품을 피하기 위해 조심스럽게 섞는다. </li></ul></li><li> 24 웰 플레이트의 각 웰에 1 mL를 넣고 37 ℃에서 1 시간 동안 배양 젤이 설정할 수 있도록 가습 분위기에서 C를 °. 1 시간 후 젤 위에 1 ML DMEM (10 % FCS) / 과잉 추가 하룻밤 인큐베이터로 돌아갑니다. </li><li> 다음 날, 젤 및 5 × 5 CRC 상피 세포를 포함하는 미디어의 판 1 ㎖의 상단에서 대기음 매체. </li></ol> 겔 코팅 된 나일론 시트의 제조 : <ol start="5"><li> 1.5 cm 크기의 멸균 나일론 시트는 사전에, 1.5 cm를 멸균 x 다음 준비합니다. </li><li> 무균 핀셋을 사용하여, 10cm 배양 접시 (통상 4 나일론 시트 인 접시 당 수용 할 수있다)에 나일론 시트의 필요 개수를 배치. </li><li> 다음과 같은 순서로 얼음 젤 메이크업 (250 μL의 총볼륨은) 나일론 장에 필요합니다 : <ul><li> 쥐 꼬리 콜라겐의 7 권 </li><li> 필터링 10 배 DMEM의 1 볼륨 </li><li> FCS의 1 볼륨 </li><li> 1 DMEM의 볼륨 10 % FCS / 공급 과잉 </li></ul></li><li> 액이 황색 인 경우,이 솔루션은 분홍색으로 변할 때까지 50 μL 씩 분주 0.1 M NAOH를 추가하여 중화. </li><li> 각 나일론 시트에이 솔루션을 250 μl를 추가하고 37 품어 ° C 젤이 설정할 수 있도록 30 분 동안 5 % CO 2의 가습 된 분위기에서. </li><li> PBS에 1 % 글 루타 알데하이드 (glutaraldehyde) 솔루션을 구성한다. 젤이 설정되면 각 10cm 배양 접시에 10 mL를 넣고 1 시간 동안 4 ° C에서 알을 품다. </li><li> 세척 나일론 시트는 PBS와 DMEM에 1X (10 % FCS) / 글루의 3 배, 4 ℃에서 DMEM / 글루에서 하룻밤 배양 </li></ol> 침공 강철 격자에의 Organotypic 젤을 올리기 : <ol start="12"><li> 삼각대 형성에 스테인리스 강판을 접어 발판 그리드를 미리 준비합니다. 사용 전에 압력솥.</li><li> 여섯 웰 플레이트의 각 웰에 철강 격자를 배치 에탄올 소독 집게를 사용합니다. </li><li> 각 그리드에, 최상위 콜라겐면 젤 코팅 된 나일론 시트를 놓습니다. </li><li> 조심스럽게 에탄올 소독 주걱을 사용하여 제기 나일론 시트를 24 웰 플레이트에서의 Organotypic 젤을 전송합니다. </li><li> 나일론 시트 매체와 접촉하지만 침수되지 때까지 10 % FCS와 보충 DMEM 10 % FCS / 공급 과잉으로 잘 입력합니다. 매체의 Organotypic 젤을 만지지 않도록하십시오. </li><li> 2 일마다 미디어를 교체, 5 % CO 2의 습한 분위기에서 37 ° C에서 14 일 동안 배양한다. </li></ol> 3.의 Organotypic 고정 <ol><li> 우물에서 나일론 시트를 포함한 전체의 Organotypic를 제거하고 집착 필름에 배치합니다. </li><li> 의 Organotypic 깨끗한 일회용 메스를 사용하여 나일론 시트를 양분하고 실온에서 24 시간 동안 포름 알데히드의 두 반쪽을 고정합니다. </li><li> 70 %의 에탄과 포름 알데히드를 교체올 24 시간 후 전에, 파라핀에 삽입 구획 및 염색에 하룻밤 둡니다. </li></ol> 침략적인 여백의 4. 레이저 미세 절제 <ol><li> 막 상에 10 ㎛ 두께로 섹션 슬라이드를 장착. </li><li> 1 분 동안 크실렌을 적용하여 Deparaffinize 섹션은​​ 다음 추가로 1 분 동안 크실렌을 제거하고 75 %의 에탄올에 고정합니다. </li><li> 1 분 0.125 % 크레 실 바이올렛 용액을 에탄올과 얼룩을 제거합니다. 크레 실 바이올렛 상피 세포를 강조하고 기질에서 쉽게 차별을 허용합니다. </li><li> 크레 실 바이올렛을 제거하고 100 % 에탄올로 세척하기 전에 추가로 1 분 동안 100 % 에탄올을 적용합니다. 30 분 동안 공기 건조에 슬라이드를 허용합니다. </li><li> 선택 플랫폼 (예를 들어, 라이카 AS 시스템)를 사용하여 레이저 미세 절제를 실시한다. </li><li> 현미경 무대에 얼굴을 아래로 microdissected 할 스테인드 섹션 유리 슬라이드를 놓습니다. </li><li> 컬렉션 카세트에 0.5 ㎖ microcentrifuge 관을 장착하고 50 μL의 추가microdissected 조직을 수집 할에 대한 상한에 세포 용해 버퍼 (예 : DNA, RNA 또는 단백질의 용해 버퍼). </li><li> 직시 하에서, 그 조직을 식별하기 위해 조이스틱을 사용하고, 소프트웨어 인터페이스를 사용하여 종양 침윤 전면 microdissected 할 셀을 강조 더러워 유기 부분을 주석. </li><li> 강조 표시된 부분을 잘라해야 모두 발사 레이저를 지시하고 microcentrifuge 관의 뚜껑에 microdissected 조직을 추진. </li><li> 일단 충분한 자료를 수집 한, 컬렉션 카세트를 꺼내 microcentrifuge 관을 닫고 튜브의 바닥에 용해 버퍼를 그리는 부드럽게 회전. </li><li> 미세 절제가 완료 될 때까지 얼음에 샘플을 놓습니다. 적절한 방법으로 처리 샘플은 관심의 분석을 추출 할 수 있습니다. </li></ol>

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저자는 그 잠재적 인 충돌을 공개하지 않습니다.

여기에 우리가 특별히 분리하고 상피와 기질 세포의 3 차원 공동 문화 구조의 전이성 진행의 초기 단계에서 암 관련 기질을 공격 할 수있는 능력을 획득 한 종양 세포의 특성을하는 방법을 설명합니다. 생체 인간의 대장 섬유 아 세포를 포함하는 합성 기질과 나란히 CRC 상피 세포로 구성된 공동 문화 모델은 3 차원에서 CRC의 침략을 연구하는 데 사용되었습니다. 생?...

의 Organotypic 공동 배양하는 중요한 세포 외 기질 구성 요소 1-3를 포함하는 콜라겐 겔에 악성 상피 세포와 기질 세포를 병치하여 3 차원 생체 내 종양 미세 환경을 재구성에 도움 조직 공학적 모델입니다. 주로 종양의 침략을 측정하는 방법으로 잉태, organotypics 생체 내 동물 모델에 대한 의존도를 줄이고, 이러한 침입 세포가 단 분산 상태 2-4로 인위적으로 강제되는없이 Transwell 분석과 같은 다른 생체 기술의 단점을 피할 수 있습니다. 이러한 섬유 아세포와 같은 이러한 모델에서 기질 세포의 포함은, 악성 침윤과 전이 3-4 조절에 종양 미세 환경의 필수적인 역할을 반영하지만 기질의 표현형 이질성은하다의 Organotypic의 생리 학적 관련성을 최대화하기 위해 모델, 장기 별, 해부학 적으로 정확한 생체가능하면 3,6 섬유 아 세포가 포함되어야한다. Organotypics 종양 및 간질 세포 사이의 상호 작용을 연구 할 수있는 다양한 플랫폼이며, 점점 화학 억제제의 종양 침윤에 미치는 영향 및 표적 유전자 변경 7,8를 검사하는 새로운 방법으로 사용된다. 침략 종양 마진을 공부하는 것은 특히 흥미로운 전망이다. 의 Organotypic 모델 확립 결장암 (CRC) 세포주는 일반적으로 잘 성층 상피 층을 생산하고, 단면에서, 세포 외 기질 (ECM)를 침입 할 수있는 능력을 획득 한 세포가 쉽게 식별 할 수있다. 레이저 미세 절제를 사용하여 분리를 공부하고이 세포를 추출 종양 및 생체 내에서 종양 관련 기질 세포의 설립 마이크로 지형 이성의 빛으로, 대장 암 전이의 기원에 관한 중요한 생물 학적 통찰력을 보여줄 수 있습니다, 어떻게보다 효율적으로 될 수있다 대상. 나는N 다음과 같은 방법론은 조직의 동의서를 작성 샘플을 제공하고, 연구를 기증 한 모든 환자는 지역 연구 윤리위원회 기관에 의해 승인되었습니다.

<table><tbody><tr><td>Colorectal cancer cell lines - example shown SW480</td><td>ATCC</td><td>ATCC CCL-228</td><td></td></tr><tr><td>Collagen</td><td>BD Biosciences</td><td>354265</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354234</td><td></td></tr><tr><td>Nylon membrane</td><td>Merck Millipore</td><td>VVLP01300</td><td></td></tr><tr><td>Metal grid</td><td>The Mesh Company</td><td>WSS20-A4</td><td>themeshcompany.com</td></tr><tr><td>Laser microdissection platform</td><td>Leica Microsystems</td><td>Leica AS LMD</td><td></td></tr><tr><td>Membrane mounted slides</td><td>Molecular devices</td><td></td><td></td></tr><tr><td>Cresyl Violet</td><td>Merck Millipore</td><td>1052350025</td><td></td></tr></tbody></table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

대장 암에 침입 (CRC) 세포는 자신의 여동생 세포에서 무료로 휴식 기질에 침투하고, 세포 외 기질 (ECM)를 개조 할 수있는 능력을 획득했다. 세포의 표현형이 서로 다른 그룹의 생물학을 특성화하는 것은 실질적으로 전이 놓기 동안 초기 사건에 대한 우리의 이해를 향상시킬 수 있습니다. 종양의 침략 악성 상피 암 관련 기질 사이의 양방향 상호 작용에 의해 촉진 역동적 인 과정이다. 우리는 공동의 문화와 레이저 마이크로 해부 기술의 Organotypic을 결합했다 종양의 기질 인터페이스에서 세포 고유의 반응을 조사하기 위해. 이러한 섬유 아세포와 같은 주요 기질 성분 3 dimentioanally 암 상피 세포와 공동 배양 된의 Organotypic 모델, 가까운 산도에서 공부하는 침략과 암 - 기질 상호 작용을 가능하게 높은 처리 가능한 실험 도구입니다ysiological 조건. 레이저 미세 절제 (LMD)는 미크론 수준의 정밀도를 가진 종양 조직 내에서 다양한 계층의 외과 절개 및 추출을 수반하는 기술입니다. 게놈 transcriptomic과 성적인 프로파일과 이러한 기술을 결합함으로써 우리는 종양 세포를 침입 및 간질 조직을 주변의 분자 특성에 대한 깊은 이해를 개발하고, 그렇게함으로써 잠재적으로 새로운 바이오 마커 및 CRC에있는 약물 개발을위한 기회를 공개하는 것을 목표로하고 있습니다.

우리는 CRC 세포주 및 간질 세포의 여러 조합에 상기 방법을 적용했다. 여기에 제시 한 예는 인간의 기본 생체 대장 섬유 아세포와 상피 CRC SW480 세포주이다. 3 차원의 공동 문화는 침략 종양 전면 세포 사이의 차등 발현의 miRNAs의 비교 현미경과 레이저 캡처 미세 절제 (그림 2)을 실시한다 (그림 1), 건설 및 마이크로 RNA (miRNA의) 프로파일 (그림 3)에 의해 ?...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

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

합성, 조직 공학 대장 암 모델에서 레이저 microdissected 세포 및 기질의 분자 프로파일은 침습적 전방 및 암 관련 기질 세포에서 종양 세포 간의 계면에서 독특한 생물학을 특성화하고 해부 소설과 같은 처리 방식을 나타낸다.

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

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

molecular-profiling-invasive-tumor-microenvironment-3-dimensional

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

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

11.0K 조회수.  University of Southampton School of Medicine.  합성, 조직 공학 대장 암 모델에서 레이저 microdissected 세포 및 기질의 분자 프로파일은 침습적 전방 및 암 관련 기질 세포에서 종양 세포 간의 계면에서 독특한 생물학을 특성화하고 해부 소설과 같은 처리 방식을 나타낸다. 

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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

종양의 4D 라이브 세포 이미징을위한 MAME 모델 : Microenvironment 상호 작용 영향을 미치는 악성 진행

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

연구

의학

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D 스페로이드 모델은 시험관 내 암 관련 섬유아세포 분화 및 침입을 위한 보다 생리적 시스템으로

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

암 연구학

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

종양-기질 상호 작용 및 약물 발견을 연구하기 위한 새로운 기질 섬유아세포 변조 3D 종양 스페로이드 모델

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

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

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

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

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

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

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 ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

3D 다세포 폐 종양 스페로이드에서 분리된 암 관련 섬유아세포의 미토콘드리아 건강 평가

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

준비 및 분석<em&gt; 체외</em&gt; 3 차원 유방 암 서로 게이트

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

생체공학

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 ...

암 생물학의 3D 모델: 헤테로스페로이드의 침입 역학

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

저자 스포트라이트: 두경부암 진행 중 암과 면역 세포 사이의 누화 해독

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및

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