Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
Uploaded: 2018-01-08T13:00:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

이 작품은 Fondo de Apoyo Investigación, 병원 Infantil 드 멕시코 페데리코 고메스 (프로젝트 번호 그-2014-053) 라와 CONACyT FONSEC SSA/IMSS/ISSSTE 프로젝트 번호 233061 Ezequiel M. 푸 엔 테 스-Pananá에 의해 지원 되었다. 프란 산체스 나 프로그램이 드 Doctorado en Ciencias Biomédicas, 대학 나시오날 Autónoma 드 멕시코 (UNAM)과 수신된 친목 231663 CONACYT에서에서 박사 학생입니다. E-S 나, 사회 보장 (IMSS)의 멕시코 연구소에서 제공 하는 금융 지원 인정.

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저자는 그들이 하지 않아도 어떤 상충 선언 합니다.

3D 공간 구조에서 상피 세포 성장 그리고 ECM 단백질의 상호 작용은 조직 항상성에 대 한. Monolayers (2D)에서 성장 하는 세포에 많은 암 연구 기반으로 되어, 비록 그들은 종양 형성 및 진행의 여러 측면을 이해 하는 데 중요 한 되어, monolayers ECM에 대 한 셀에 부과 하는 특징을 정리 하지 않습니다. 인스턴스: 확산, 접착 종속 세포 생존, 꼭대기 basolateral 극성, 개장 하는 ECM, 세포 분화, 등 제한 중?...

종양 생물학은 이전에 생각 보다 훨씬 더 복잡 한. 종양 세포는 억제 되지 않는 확산 셀;의 단순한 대량 보다 더 많은 보여주는 증거를 장착 오히려, 다른 종양 약 세포 높은 조직과 종양1내에서 계층 구조를 표시 하는 다른 기능을 수행 같다. 종양 세포는 또한 비 변형 세포와의 친밀 한 커뮤니케이션에서: 대 식 세포, 섬유 아 세포, 림프 톨, adipocytes, 내 피 세포, 다른 셀 들 모두에 몰입 하는 비 계 단백질 및 다 당 류는 ECM을 구성 하는. 수많은 직접 및 간접적인 상호 작용 변환 및 비 변형 세포 사이 및 질병 결과2,3에 강력한 영향을 미치는 ECM으로 설정 됩니다. BrC의 특정 한 경우에 그것은 특히 Tam, 고려는 Tam 발견 되었습니다는 종양의 진화에 중요 한 역할을 증가 하는 전이의 위험과 질병 재발4의 BrC 셀의 통신을 해 부하는 것이 중요 ,5.
내부 종양 상호 작용 및 그들의 가능한 결과 분석, 새로운 3D 생체 외에서 방법을 개발 되었으며, 훨씬 더 복잡 한 microenvironment를 제공 하는 ECM 추출 물의 사용에 따라 종양 생물학, 비교에서의 현실에 가까운 기존의 단층 세포 배양 세포 성장 플라스틱에 첨부. 피터슨와 Bissell6 아의 첫 번째 모델을 제공 하 고 악성 유 방 상피 세포 laminin 풍부한 지하실 멤브레인에 교양 아 인간을 차별 하는 3D organotypic 구조를 설명 하는 첫번째 이었다 그들의 악성에서 유 방 상피 세포입니다. 10 년 뒤, 모델 Debnath, Muthuswamy에 의해 개발 및 브루 게7,,89 선의 acini의 악성 변환 하는 동안 손상 하는 생물 학적 경로 명료 하 게 하는 귀중 한 도구 제공 등 통제 확산, 장애인된 셀 분극의 증거 및 anoikis, 셀 저항 결과로 acini 루멘으로 꽉 접합 단백질의 delocalization 인해 큰 acini 형성으로 프로그램 된 세포 죽음의 종류를 발생 앵커리지-종속 셀 때 그들은 주변 ECM에서 분리. Sameni, Jedeszko, 및 슬론의 모델은 밀접 하 게 침입, 종양 암10,,1112의 또 다른 중요 한 특성에 관련 된 세포에 의해 이미징 분해 활동에 집중 했다. 섞인 다른 형광 침묵 단백질 기질 단백질 행렬에 의존 하는이 모델 (DQ 젤라틴, DQ-콜라겐, 그리고 DQ 콜라겐 IV), 어떤 형광 신호에는 콜라겐의 분해 저하의 지표. 3D 모델 변형 비 둘 다의 줄기 세포 속성과 셀의 집계, spheroids 라고 하는 종양 세포를 공부 하는 데도 사용, ECM 같은 단백질 세포 분화, 비대칭의 메커니즘에 대 한 질문 또는 정지에 경작 될 수 있습니다. 세포 분열, 세포 준수, 및 세포 운동 성13,14. 침공 분석 실험 종양의 본질적인 공격 성과 침략 과정15중 chemoattractants로 봉사 하는 분자의 식별의 테스트 수 있습니다. 전반적으로, 3D 모델의 체 외에서 세포 배양 정상 및 종양 조직 morphogenesis 더 밀접 하 게 반영 하는 저렴 한 다양화를 나타냅니다.
우리 인간의 상업 BrC 셀 라인 알려진된 공격적 잠재력 (luminal 및 트리플-네거티브 형식) 및 기본을 사용 하 여 상기 모델7,,1011에 따라 3D 셀 공동 문화 시스템 설계 셀 explanted BrC 환자에서. 우리가 먼저 적극적인 비 (MCF-7) 또는 공격적 (MDA-MB-231) BrC 셀 U937 monocytes 기질 추출 (ECME)와 공동 경작 했다 모델 개발-직접 셀 상호 작용을 허용 하는 3D 시스템을 기반으로. 이러한 공동 문화 어떻게이 두 세포 계보 간 통신 암 공격적인 행동에 관련 된 유전자의 세트의 녹음 방송에 영향을 결정 하기 위해 사용 되었다. 2 cyclooxygenase (COX-2) 대 본의 상당한 증가 하는 제품 중 하나는, 스타 글란 딘 E2 (PGE2), 암 진행에서 염증의 역할을 강조 하는 발견의 생산 증가와 동시에 관찰 되었다. MMP의 증가 전사 또한 적극적인 MDA-MB-231 셀 U937 monocytes DQ 콜라겐 IV 포함 된 문화에 함께 공동 경작 했다 때 큰 콜라겐 베이스와 상관 하는 관찰 되었다. 메모의, 우리의 공동 문화는 세포 세포 상호 작용 메커니즘 콜라겐 저하에 필요한 가정을 지원 하지 않았다. 그것은 오히려 두 세포 계보 간 통신 분 비 분자에 의해 중재 했다 제안 했다. 또한, 이러한 공동 문화 분석 실험에서 수확 supernatants 선의 acini MCF 10A 세포 변형 비13에 의해 형성 된 무질서 수용 성 요소 포함. 그것은 발견 하는 적극적인 고 기본 BrC 세포 분 비 상승 monocyte 혈 분자 MCP-1 GM-CSF와 RANTES의 수준. 따라서, 우리는 세포 세포 세포 상호 작용을 방지 하기 위해 셀 문화 삽입에서 분리 되었다 3D 문화 설명. 이러한 문화는 BrC 세포, monocytes 사이 간접 통신을 해결 하기 위해 사용 되었다. 이 분석 실험, 비 공격적이 고 적극적인 상업 BrC 셀 라인 및 기본 BrC 셀, 인간 monocytes의 세 가지 유형: 상업 U937 및 THP-1 셀, 그리고 기본 monocytes (PMs) (모두 건강 한 기증자의 주변 혈액에서 분리 monocytes는 활성화 되지 않은 상태에서 사용 했다) 사용 되었다. 염증 성 cytokines 일리노이-1β 및 IL-8의 증가 농도 공동 문화에 농축 될 관찰 되었다. 마찬가지로, MMP-1, MMP-2, 및 MMP 10 BrC 셀 monocyte 공동 문화, 그리고 이렇게 강화 이전 결과14에 증가 또한 했다 발견 했다. 이 원고에서 기본 BrC 세포 격리 및 3D 문화에서 테스트 포인트 워크플로 대표적인 결과 함께 제공 됩니다. 이 작품 구성 종양 생물학의 특정 측면이 3D 셀 시스템 생체 외에서 제공 하는 중대 한 잠재력의 좋은 예.

<table><tbody><tr><td>U937</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-1593.2</td><td>Monocytic cell line/histiocytic lymphoma&amp;nbsp;</td></tr><tr><td>THP-1</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>TIB-202</td><td>Monocytic cell line/acute monocytic leukemia&amp;nbsp;</td></tr><tr><td>MCF-10A</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>CRL-10317</td><td>Non-transformed breast cell line</td></tr><tr><td>MCF-7</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-22</td><td>Breast Cancer Cell line</td></tr><tr><td>T47D</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-133</td><td>Breast Cancer Cell line</td></tr><tr><td>HS578T</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-126</td><td>Breast Cancer Cell line</td></tr><tr><td>MDA-MB-231</td><td>American Type Culture Collection ATCC&amp;nbsp;</td><td>HTB-26</td><td>Breast Cancer Cell line</td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO BRL Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>11964-092</td><td>4.5 g/L glucose</td></tr><tr><td>DMEM/F12</td><td>GIBCO BRL Life Technologies</td><td>11039-021</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>GIBCO BRL Life Technologies</td><td>15240-062</td><td>100 U/mL penicillin, 100 &amp;micro;g/mL streptomycin, and 0.25 &amp;micro;g/mL Fungizone</td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO BRL Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Horse serum&amp;nbsp;</td><td>GIBCO BRL Life Technologies</td><td>16050114</td><td></td></tr><tr><td>0.05% Trypsin-EDTA 1X</td><td>GIBCO BRL Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>PBS 1X (Phosphate Buffered Saline</td><td>GIBCO BRL Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>AF-100-15 (1.00mg)</td><td></td></tr><tr><td>Insulin</td><td>SIGMA-ALDRICH</td><td>I1882-100MG</td><td></td></tr><tr><td>Hydrocortisone</td><td>SIGMA-ALDRICH</td><td>H088-5G</td><td></td></tr><tr><td>Cholera toxin Vibrio cholerae</td><td>SIGMA-ALDRICH</td><td>C8052-1MG</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Corning Inc</td><td>356237</td><td>Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&amp;deg;C until use at 4&amp;deg;C</td></tr><tr><td>GM-CSF</td><td>PeproTech</td><td>300-03</td><td></td></tr><tr><td>MCP-1</td><td>PeproTech</td><td>300-04</td><td></td></tr><tr><td>RANTES</td><td>PeproTech</td><td>300-06</td><td></td></tr><tr><td>IL-8</td><td>PeproTech</td><td>200-08</td><td></td></tr><tr><td>IL-1&amp;beta;</td><td>PeproTech</td><td>200-01B</td><td></td></tr><tr><td>Crystal-violet</td><td>Hycel M&amp;eacute;xico</td><td>541</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>P6148</td><td></td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Corning Inc</td><td>3422</td><td>6.5 mm diameter, 8 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Transwell permeable supports (inserts)</td><td>Thermofisher Scientific</td><td>140620</td><td>6.5 mm diameter, 0.4 &amp;micro;m pore size/24-well plates&amp;nbsp;</td></tr><tr><td>Monocyte Isolation Kit II Human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-153</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td><td>EMD Millipore</td><td>HCYTOMAG-60K</td><td></td></tr><tr><td>Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td><td>Luminex Corporation</td><td>40-072</td><td></td></tr><tr><td>Lab-Tek chamber slide with cover (8 well)</td><td>Nalge Nunc International</td><td>177402</td><td></td></tr><tr><td>Glass bottom microwell 35mm petri&amp;nbsp; dishes</td><td>MatTek Corp.</td><td>P35G-1.5-14-C</td><td></td></tr></tbody></table>

analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system

세포 외 기질 (ECM)에 포함 된 정상 및 종양 약 상피 세포 속속들이 조 혈 및 비 조 혈 세포, 따라서 크게 영향을 미치는 정상 조직의 항상성 및 질병 결과와 통신 합니다. 유방암, 종양 관련 된 대 식 세포 (Tam) 역할을 중요 한 질병의 진행과 전이, 재발; 따라서 질병을 제어 하는 것이 중요이 monocyte chemoattraction 종양 microenvironment 및 종양 세포와 그들의 상호 작용의 메커니즘을 이해. 여기, 우리는 인간 유방암 (BrC) 세포 및 인간 monocytes의 3 차원 (3D) 공동 문화 시스템에 대 한 자세한 설명을 제공합니다. BrC 셀 생산 활성화에 규제의 높은 기초 수준, 정상적인 T 세포 표현 (RANTES) 분 비, monocyte chemoattractant 단백질-1 (MCP-1), 그리고 granulocyte macrophage 식민지 자극 인자 (G-CSF), monocytes, 공동 문화에 프로 염증 성 cytokines 인터 루 킨 (IL)-1 베타 (IL 1β) 및 IL-8 매트릭스 metalloproteinases (MMP)-1, MMP-2, 및 MMP-10 풍성 하 게 했다. 이 종양 기질 microenvironment 승진 anoikis MCF 10A 3D acini 같은 구조, monocytes의 chemoattraction 그리고 적극적인 BrC 셀의 침공에 저항. 여기에 제시 된 프로토콜 내 종양 커뮤니케이션을 공부 하는 저렴 한 대안을 제공 하 고 3D 셀 시스템 생체 외에서 심문 종양 관련 종양 생물학의 특정 기능을 제공 하는 중대 한 잠재력의 예 침략입니다.

BrC 셀 3D 문화에서의 형태소 분석:
낮은 높은 밀도에서 3D 문화에서 성장 하는 기본 BrC 셀의 형태는 5 일 동안 연구 했다. 첫 번째 48 h 동안 세포는 ECME를 준수 하 고 낮은 밀도 유지. 이 시간이 시점에서 그것은 명확 하 게 감상할 수 있습니다 세포는 길쭉한 스핀 들-모양, 명백한 셀 연락처를 표시 하지 않고 두 개 이?...

Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

(ECME) 추출 Monocytes 그리고 기본 유 방 암 세포는 세포 외 기질 간의 통신을 분석-기반 3 차원 시스템

여기, 우리는 기본 유 방 암 세포 뿐만 monocytes 그리고 콜라겐 저하, 면역 세포 모집, 셀 결과 함께 그들의 직접/간접적인 상호 작용 연구로 형태를 분석 하는 3 차원 문화 방법 설명 침공, 그리고 암 관련 된 염증의 승진.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

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Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

10.9K 조회수.  Hospital Infantil de México Federico Gómez. 여기, 우리는 기본 유 방 암 세포 뿐만 monocytes 그리고 콜라겐 저하, 면역 세포 모집, 셀 결과 함께 그들의 직접/간접적인 상호 작용 연구로 형태를 분석 하는 3 차원 문화 방법 설명 침공, 그리고 암 관련 된 염증의 승진.

비디오: Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

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

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

미생물 시스템을 사용하여 인간 유방 조직에서 유방암 모델링

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

암 연구학

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

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

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

배양 인간의 대퇴골 조직 외식하는 방법은 전이성 틈새의 유방암 세포 집락을 연구하기 위해

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

<meta charset="utf8"></head><body>3D 콜라겐 매트릭스 설정에서 대식세포-종양 스페로이드 상호 작용 검사

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>대식세포-종양 스페로이드 동시 침습 분석

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

대식세포-종양 스페로이드 동시 침습 분석

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

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 ...

(ECME) 추출 Monocytes 그리고 기본 유 방 암 세포는 세포 외 기질 간의 통신을 분석-기반 3 차원 시스템

요약

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이 비디오의 챕터

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

미생물 시스템을 사용하여 인간 유방 조직에서 유방암 모델링

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

준비 및 분석

배양 인간의 대퇴골 조직 외식하는 방법은 전이성 틈새의 유방암 세포 집락을 연구하기 위해

대식세포-종양 스페로이드 동시 침습 분석

대식세포-종양 스페로이드 동시 침습 분석

대식세포-종양 스페로이드 동시 침습 분석

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