와 제임스 Neiswender, 순영 관이 작업에 사용 plasmids의 선물의 고 (故) 치 빈 치엔; 항체 염색법과 도움 제임스 리스터와 데이빗 Raible 우리는 그의 연구실이 기술이 처음 개발에서 박사 레너드 I. Zon 감사 microinjection 비디오. 이 작품은 CJC에 NIH 보조금 R00AR056899-03가 추진하는 사업

1. 흑종의 발병 조절을위한 검사 <ol><li> PCR 관심의 유전자의 전체 길이 열려있는 독서 프레임 (GOI)를 증폭하고 BP clonase II (Invitrogen)를 사용하여 pDONR 221에 ​​recombining에 의해 게이트웨이 중간 항목 클론을 만들 수 있습니다. miniCoopR 벡터 (그림 1A)에 mitfa 프로모터에서의 관심 유전자를 삽입 재조합 p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 miniCoopR 5 Multisite 게이트웨이 기술 (Invitrogen)를 사용합니다. </li><li> 이전 7에 설명 된대로 mitfa (LF) triply homozygous zebrafish의 배아, p53의 (만약에) : 한 셀 TG (BRAF V600E mitfa)에 25 picograms Tol2 transposase의 mRNA와 함께 각 클론의 25 picograms를 삽입. 28.5에서 주입 배아를 배양 ° C. 24 hpf에있는 죽은 태아를 제거합니다. </li><li> 해부 microscop에 주입 배아를 포함하는 페트리 접시를 배치하여 72 hpf에서 구출 melanocytes과 유전자 변형 동물을 선택흰색 배경 (그림 1A)에 대한 입사광에 따라 5. </li><li> 이전에 8에 설명 된대로 네 dpf에서 zebrafish 시설의 보육의 3 L 탱크에 동물을 전송합니다. </li><li> 2개월에서 4mm 2 (그림 1A)보다 큰 melanocyte 구조의 적어도 하나의 공간이있는 동물을 선택합니다. 72 hpf에서 melanocyte 구조의 정도, 2 개월 melanocyte 구조의 정도 사이에 강한 상관 관계가 있습니다. melanocyte 구조와 태아가 72 hpf로 선택하는 경우, 그들 중 대부분 (~ 80 %) 2 개월 4mm 2보다 큰 melanocyte 구조의 적어도 하나의 영역을 갖게됩니다. </li><li> 화면 종양의 존재 (그림 1B)에 대한 주간 선택한 동물. histopathologic와 morphologic 변경 사항을 사이에 강한 상관 관계가 있습니다. 양성에서 악성으로 전환이 병변은 동물의 표면을 제기 될 morphologic 변경으로 인정 받고 있습니다. 종양 베어링 애니 마을 분리연구 있나요. </li><li> 종좌표에 자리표와 %의 흑색 증이없는 생존의 주 나이가 흑색 증 - 무료 생존 곡선을 그립니다. 관심있는 유전자를 표현하는 melanocytes가있는 동물은 동물을 제어하는 비교됩니다 melanocytes에서 명시 적 EGFP (강화 된 녹색 형광 단백질) (그림 1C). miniCoopR-EGFP를 주입 동물에 대한 중간 종양의 증상은 약 18 주입니다. 두 곡선이 로그 순위 테스트를 사용하여 통계적으로 다른 있는지 여부를 확인합니다. </li></ol> 2. 종양 침략 분석 <ol><li> hindbrain과 등쪽 지느러미 (그림 2A)의 앞쪽에 경계의 뒤쪽 경계 사이의 지역에서, dorsally 종양을 개발하는 절연 zebrafish를 선택합니다. </li><li> 흑색 증의 증상은 메사추세츠 의과 대학 기능 대학에서 안락사에 대한 미국의 동물 의료 협회 패널에 의해 지정된 및 승인 지침에 따라 물고기를 안락사시켜야 2 주 후에titutional 동물 관리 및 사용위원회 (IACUC). 아가미의 움직임이 멈출 때까지 특히, 생선은 0.6 MG / ML tricaine와 함께 그릇에 배치됩니다. </li><li> 메스 생선의 복막 캐비티에 절개를 확인하고 고정에 대해 4 ° C에서 밤새 4 % paraformaldehyde에있는 물고기 (PFA)를 넣습니다. </li><li> 4에서 하루 아침에 0.5 M EDTA (에틸렌 다이아 민 테트라 초산)와 함께 치료 ° C가 decalcify합니다. </li><li> 밤 60시 2 시간 각 2 회 ° C.를 1 시간에 크실렌 3 회와 분명 1 시간에 탈수하고 파라핀으로 처리 카세트에 포르말린으로 고정 </li><li> 몰드에 파라핀의 조직을 삽입하고 30 분 ~ 1 시간을 위해 더욱 강화 할 수 있습니다. </li><li> 5 μm 섹션, 하나마다 50 μm하십시오. 섹션은 가로와 깊은 침략의 요점을 식별 할 수 있도록 전체 병변을 통해해야합니다. hematoxylin 및 eosin의 착색에 따릅니다. </li><li> 종양 세포가 collagenous 지층 compactum 밖에 남아 있는지 여부를 측정하여 종양 침략을 평가 등쪽에 침략근육은 나 척추 (그림 2B, C)에 더 침략. 샘플의 두 집합 사이의 통계적 차이를 확인합니다. </li></ol> 3. 규모 Melanocytes의 항체 염색법 <ol><li> 80 ML 0.1 M 오세영 2 HPO 4, 20 ML 0.1M 아니 2 PO 4 PO4 버퍼를 확인합니다. 산도는 7.3 있는지 확인하십시오. 8.0 g의 자당, 0.15 ML 0.2M CaCl 2, 90 ML 0.2M PO 4 버퍼, 산도 7.3을 포함하는 2 배 수정 버퍼를 확인합니다. 필요는 NaOH 또는 HCL과 7.3로 pH를 조정한다면 PO 4 버퍼 100 ML까지합니다. </li><li> 300 MG 고체 PFA까지 무게 microcentrifuge 튜브에 추가합니다. PFA 4.5 X 질량, MG에서 (예를 들면 450 UL 5 MM NaOH 100 MG PFA까지)와 같은 볼륨에 5 MM NaOH를 추가합니다. 60-70에서 열 ° C PFA가 해산 될 때까지 흔들림 가끔있는. 나머지 입자를 스핀 다운 20 % PFA 표면에 뜨는을 복구합니다. 신선한 때마다 확인합니다. </li><li> 1X Fi 접속 설비를 포함하는 고정 솔루션을 준비X 버퍼와 4퍼센트 PFA. </li><li> 0.17 MG / ML tricaine에서 물고기를 마취. </li><li> 날카로운 미세 스쳐 집게를 사용하여 등쪽의 비늘은 꺽을 해부 현미경으로 입사 광을 사용하여 규모의 melanocyte 함유 절반 (그림 3A)를 손상하지 돌봐. 직접 한 ML의 고정 솔루션과 실온에서 알을 품다,에 포셉 곳에서의 비늘가 도는 동안, ≥ 2 시간에. 물고기 물에 물고기를 전송하고 성공적인 복구를 확인하기 위해 생선을 모니터링 할 수 있습니다. </li><li> 표백 색소 melanocytes는 실온에서 5 분에 PBST (1X PBS 산도 7.4, 0.1 % 트리톤 X-100) 2 번에 고정 비늘을 씻어합니다. DH 2 O. 5 개 ML에 0.4 ML 10% 코와 0.15 ML 30% H 2 O 2를 포함 한 ML을 추가 갓 만든 표백 솔루션 튜브가 열려 파열에서 가스를 방지하기 위해 parafilm이나 모자를 자물쇠에 놔. 안료는 (그림 3B, C) ​​(10 분 전형적인) 사라질 때까지 표백 계속합니다. 온도는 실온에서 5 분 동안 PBST 네 번에 씻어erature. </li><li> 1X PBS 산도 7.4, 0.2 % 트리톤 X-100, 2 밀리그램 / ML BSA, 1 % DMSO, 0.02 % 할머니 3 2% 양 혈청으로 구성 블록 솔루션에 적어도 30 분을위한 블록. HRP 반응을 개발하면 할머니 3을 둡니다. </li><li> 실온에서 하룻밤 블록 솔루션에 기본 항체를 품다. 당신이 그들을 침수 유지하기 위해 샘플을 통해 솔루션의 적어도 400 μl이 필요합니다. </li><li> 상온에서 PBST에서 30 분의 비늘에게 3 번 씻으십시오. </li><li> 2 시간에서 실온에서 블록 솔루션 야간에 차 항체를 품다. </li><li> PBST + 0.1 μM의 DAPI로 10 분에 청바지. </li><li> PBST로 5 분에 3 번 씻으십시오. </li><li> 비늘의 오목면이 슬라이드 밑에 직면 있도록 vectashield 한 방울의 유리 슬라이드에 비늘를 장착하고 이상 유리 커버 전표를 놓습니다. 투명 매니큐어와 슬라이드의 가장자리를 밀봉하고 형광 현미경 (그림 3D, E, F, G에서 슬라이드를 관찰 </strong&gt;). </li></ol> 4. 이식 검정 <ol><li> 이식 10 이전 감마 방사선의 25 GY 일일를 irradiating에 의해받는 2~3개월 이전 캐스퍼 9 생선을 준비합니다. 물고기는 물고기 물에 복구 할 수 있습니다. </li><li> 이전에 설명 된대로 종양 - 베어링 생선 (2.2 절)을​​ 안락사시켜야. </li><li> 종양을 잘라 5 % FBS와 0.9x PBS를 소독 약 5 ML 필터를 페트리 접시에 넣어. 면도기 솔루션 중에있는 종양를 분석. </li><li> 실온에서 근무, 단일 세포를 얻을 수있는 P1000 피펫을 씹다. 5% FBS와 필터를 소독 0.9x PBS로 볼륨 25 ML로 구성합니다. </li><li> 40 μM 메쉬 필터를 통해 필터링 할 수 있습니다. </li><li> 10 분에 1,500 RPM (453 rcf)에서 Eppendorf 5810R의 탁상 원심 분리기에 봐. </li><li> 표면에 뜨는를 제거하고 약 원하는 농도 (100mm 3 종양에 대한 10 7 세포를 가정)에 세포 현탁액을합니다. </li><li> t를 계산그는 정확한 휴대폰 번호는 hemocytometer를 사용하여 최종 사출 볼륨이 약 5 μl이되도록 세포 현탁액을 희석. 50000 세포 주입 할 경우 예를 들어, 세포 현탁액은 10,000 세포 / μl의 최종 농도로 희석해야합니다. 세포 현탁액에게 전지 거리며 걷게을 방지하기 위해 모든 몇 분을 포함하는 관을 긋기. </li><li> 2 ~ 3 배는 100 % 에탄올과 0.9x PBS와 26s 게이지 (베벨 팁) 701 N 10 μl 해밀턴 주사기를 씻으십시오. 세포 현탁액의 5 μl로 주사기를로드합니다. </li><li> 0.17 MG / ML tricaine에서 방사능받는 물고기를 마취. 젖은 Kimwipe에서의 편에 자리 물고기 (그림 4A). </li><li> 한 손으로 물고기를 안정화 약 반 hindbrain와 등쪽 지느러미 앞쪽 테두리의 뒤쪽 경계 사이의 복막 캐비티 위의 생선의 측면에 45 ° 각도로 일어나 직면하고있는 베벨로 바늘을 삽입합니다. 부드럽게 플런저를 (그림 4B) 우울. &lt;/ 리&gt; <li> 생선 신선한 생선 물에 복구하고 종양 engraftment (그림 4C)를 매일 물고기를 관찰 할 수 있습니다. engraftment가 발생했습니다 경우, 지속적인 성장과 질병 개발도 볼 수 있습니다. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

miniCoopR 방법은 zebrafish melanocytes에 대한 관심의 유전자의 표현이 가능합니다. 이 방법은 zebrafish mitfa 유전자가 세포 자율적으로 역할을한다는 사실을 활용합니다. 이러한 이유로, miniCoopR 벡터에 의해 구출 melanocytes는 minigene이 있고 물리적으로 연결된다에 관심이있는 유전자를 포함 할 확신합니다. 구출 melanocytes는 명확하게 볼 수 있으며, 단일 셀 배아로 주입 된 동물에서 얻을 수 있습니다. ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 게이트웨이 재결합 시약 </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> 인용 발명 5 </td></tr><tr><td> Mitfa 항체 </td><td></td><td></td><td> 인용 발명 5 </td></tr><tr><td> FITC 염소 안티 - 토끼 IgG 항체 </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> Vectashield </td><td> 벡터 연구소 </td><td> H-1000 </td><td></td></tr><tr><td> 캐스퍼 Zebrafish </td><td></td><td></td><td> 참조 9 </td></tr><tr><td> 701N 10 μl 주사기 </td><td> 해밀턴 / 피셔 </td><td> 14-824 </td><td> 및nbsp; </td></tr><tr><td> 40 μM 필터 </td><td> BD 팔콘 / 피셔 </td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

인간의 암의 게놈 연구는 종양 하나, 둘, 셋에서 변경 아르 유전자에 대한 풍부한 정보를 굴복했습니다. 이 연구에서 발생하는 문제는 많은 유전자가 변경된다는 것입니다, 그것은 모든 사람들이 변화하는 동안 부수적으로 발생한 그에서 tumorigenesis를 몰고 유전자 변경을 구분하기 어려울 수 있습니다. 이 구별을 그리려면이 양적 종양 계속하고 배포 할 수 있도록 종양 개시 및 기타 프로세스에 변경된 유전자의 효과를 측정 할 수 있습니다 검정을 가지고 유용합니다. 여기 흑색 증의 개시 및 유지 보수에 필요한 단계를 포함 autochthonous 종양 모델 (4)를 사용하여 zebrafish에 후보 흑색 증 조절 다수의 화면에 빠른 방법을 제시한다. 이 분석의 주요 시약 어떤 게이트웨이 재결합 카세트에 커플 mitfa melanocyte 사양 계수의 야생 형 사본을 miniCoopR 벡터, 어느 후보 멜로anoma 유전자는 5 재결합 할 수 있습니다. miniCoopR 벡터는 발기인, 오픈 읽기 프레임과 야생 형 mitfa 유전자의 3&#39;-번역되지 않은 지역을 포함하는 mitfa의 구조를 minigene 있습니다. 우리는 후보 흑색 증 조절의 전체 길이 열려있는 독서 프레임을 사용하여 구조를 할 수 있습니다. p53의 (만약에); mitfa (LF) zebrafish의 배아 :이 각각의 클론 그런 다음 단일 셀 TG (BRAF V600E mitfa)에 주입 될 수있다. miniCoopR 벡터는 Tol2로 인한 transgenesis 6 통합 melanocytes를 구출됩니다. 그들이 실제로 mitfa 구출 minigene에 연결되기 때문에, 후보 유전자는 변화와 종양으로 발전 할 일부의 구출 melanocytes에 표현됩니다. 흑색 증의 개시 및 흑색 증 세포의 특성에 후보 유전자의 효과가 흑색 증없는 생존 곡선, 침략의 assays, 항체 염색법과 이식 assays를 사용하여 측정 할 수 있습니다.

하나의 셀 TG (mitfa : BRAF V600E), p53의 (만약에), mitfa (LF) zebrafish의 배아는 흑색 증의 종양 유전자 mitfa 프로모터의 조절하에 SETDB1 5 EGFP 각을 포함하는 miniCoopR 벡터를 주입했다. melanocyte 구조와 태아가 선택되고 성숙 할 수 있었다. melanocyte 구조와 세 동물 2 개월에 4mm 2보다 더 큰이 선택되었습니다. 동물 melanomas 주 단위로 상영되었다. 성인을위한 종양 발생률...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

zebrafish autochthonous 종양 모델을 사용하여 흑색 증 조절을위한 화면으로 빠른 방법은 제공됩니다. 그것은 melanocytes에서 후보 흑색 증의 유전자의 표현을 허용 miniCoopR 벡터의 이점을 걸립니다. 흑색 증 - 무료 생존 곡선, 침략 분석, 규모 melanocytes의 항체 염색법을위한 프로토콜과 흑색 증 이식 분석을 구하는 방법이 설명되어 있습니다.

Zebrafish Autochthonous 종양 모델을 사용하여 흑종 조절을위한 검사

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

Research

JoVE Journal

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13.6K 조회수.  University of Massachusetts Medical School. zebrafish autochthonous 종양 모델을 사용하여 흑색 증 조절을위한 화면으로 빠른 방법은 제공됩니다. 그것은 melanocytes에서 후보 흑색 증의 유전자의 표현을 허용 miniCoopR 벡터의 이점을 걸립니다. 흑색 증 - 무료 생존 곡선, 침략 분석, 규모 melanocytes의 항체 염색법을위한 프로토콜과 흑색 증 이식 분석을 구하는 방법이 설명되어 있습니다.

비디오: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

연구

의학

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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

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

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

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

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

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

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

당뇨병과 신진 대사 메모리의 Zebrafish 모델

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

다낭성 신장 질환의 가능한 제브라 피쉬 모델 :의 넉다운<em&gt; wnt5a</em&gt; Zebrafish의 신장의 낭종 원인

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

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

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

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

종양 기질 상호 작용에 대한 세 가지 차원의 공동 문화 모델

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

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

포도막 흑색 종의 간전이에 대한 치료로 고립 간 관류

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

피하 종양 모델에서 종양 침투 백혈구 하위 집합의 평가

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...