저자는이 연구에서 사용 된 높은 역가의 렌티 바이러스 생산을위한 파지 - EF1aL-을 DsRed-UBC-GFP-W 벡터 및 프로토콜을 제공하기 위해 보스턴 대학에서 박사 대럴 Kotton 감사합니다. 이 작품은 .NCI P30CA046934 센터 보조금은 생체 내 이미징에서 지원 DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG 번호 57-001-53 (DMC), NCI K22CA181250 (DMC)와 R01 CA140985 (CAS)에 의해 투자되었다 조직 문화는 콜로라도 AMC의 대학에서 코어.

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The authors have nothing to disclose.

프로토콜 내에서 중요한 단계 : 시험 관내 형질 도입 동안 매체 조성물의 신중한 제어를 허용 높은 역가 렌티 바이러스 입자의 사용은 (&gt; 10 8 TU / ㎖),이 프로토콜의 성공에있어 중요한 단계이다. 고 역가 바이러스 입자의 제조를위한 다양한 방법이도 18, 19를 설명 하였지만; 이 프로토콜에서 상세히 설명한 바와 같이 제조 된 렌티 바이러스 입자를 사용 <a href="http://www.kottonlab.com">www.kottonlab.com를</a> . 암 세포의 종양의 소화와 분해를위한 부드러운 방법은 성공적인 라벨과 라벨이 종양의 성장을 위해 중요하다. 삼시간 넘어 소화 시간을 연장 또는 37 ° C의 분해 온도를 증가시키는 것은 해리 세포의 수를 증가하지만, 대부분의 종양 세포 생존 및 라벨의 성공을 감소시킨다. 수정 및 문제 해결 : </stroNG&gt; 이 프로토콜은 동적으로 간주하고 각 단계에서주의 깊은 모니터링 독특한 PDX에 대한 적응을 필요로한다. 이 표준 프로토콜은 대부분의 이종 이식 종양 잘 작동하지만, 작은 변화가 다른 PDXs의 라벨을 최적화 할 필요가있다. 예를 들어, 다량 및 기질 및 세포 외 매트릭스의 조성은 일반적으로 분리 한 후, 수득 된 종양 세포의 수율에 필요한 시간에 영향을 미칠 수있는 PDXs간에 다르다. 높은 혈관 괴사 및 종양가 큰 종양으로 인한 파편 과도한 마우스 혈액 세포로 레이블링하기 어려울 수있다. 린 + 고갈이 프로토콜에 설명 된 상피 세포는 EpCAM + 농축의 사용은 PDXs의 전달 효율을 향상시킨다. 그러나,는 EpCAM + 세포의 발현 따라서 그 표현은 각 PDX에 확인해야합니다, 심지어 상피 기원의 종양에서 변화 세포 농축을위한 방법으로 사용하기 전에. Limita기술의 TIONS : 해리 세포를 체외에서의 임시 문화의 전달은 부모 PDXs에서 근본적으로 다른 레이블 종양에 상승을 제공합니다 세포 개체군의 선택이 발생할 수 있습니다. 뉴 라미니다 제와 렌티 바이러스 입자의 사전 인큐베이션 따라서이 단계에서 도입 될 수있는 전달 바이어스를 감소 형질 도입하기 어려운 세포 개체군 21 바이러스 입자의 결합을 증가 할 것을 권장한다. 우리의 경험에 의하면, 형질 세포에 의해 재구성 된 종양은 밀접하게뿐만 아니라 조직 학적으로 부모 PDXs의 기능과 유사 할뿐만 아니라 (데이터가 표시되지 RNA의 서열) mRNA 발현의 계층 적 클러스터링 분석을 사용하여. 표지 효율 종양 충실도의 관점에서 품질 관리는 모든 통로로 각각 PDX 수행한다. PDX 라벨링 생체 내 종양 드리프트 종양의 확장을 필요로하기 때문에 반복 (P) 후에 발생할 수assaging, 전달 가능한 가장 빠른 통로에서 수행해야합니다. 기술의 중요성 : 이 프로토콜은 유방암 PDXs는 형광 bioluminescently 체외에서 표시 및 동 소성 및 전이성 종양 성장의 추적을 위해 생체 내에서 재 주입 할 수있는 방법에 대해 설명합니다. 이전 연구는 PDX-파생 organoids (18) 레이블을 비슷한 전략을 사용하고 있지만, 현재의 프로토콜은 여러 PDXs의 높은 전달 효율이 발생할 종양 소화와 라벨의 변화를 포함한다. 이 기술을 마스터 한 후 미래의 응용 프로그램이나 방향 : 안정적으로 추적 마커 (GFP, 루시퍼 라제)를 표현하고, 과발현 또는 특정 유전자를 넉다운 할 수있는 능력은 (즉, pSIH1는-H1은-copGFP-T2A-순수하지 않아 shRNA를 전달하는 데 사용할 수 있습니다)은 약 근본적인 질문에 대한 답변을 PDXs의 사용을 허용확립 된 세포주와 비슷한 방식에서 종양의 성장과 전이. 특히,이 프로토콜은 전이성 캐스케이드에 장기 정착 단계를 연구하는 실험 전이 모델에서 표시 PDXs을 사용하는 방법을 보여줍니다. 다른 장기로 전이가 쉽게 가시화 살아있는 동물의 생물 발광 영상을 이용하여 정량화하고, GFP 발현 유도 절개하는데 사용 적출 기관에서 전이를 시각화 할 수있다. 이 전이 연구를위한 PDXs의 사용을 촉진하는 강력한 도구를 나타냅니다.

외과 적 절제 종양 샘플은 직접 면역 감염된 마우스에 접목되어 환자 유래의 종양 이종 이식 (PDXs)의 개발, 표준 셀 라인 이종 이식 모델에 비해 여러 가지 장점을 제공하며, 암 연구 1,2-에서 큰 진보를 나타낸다. PDXs 유지하고 제 1 통로에서 성장한 종양의 유전 적 및 생물학적 특성에 최소한의 변경으로 연속적인 통로로 확장 될 수있다; 보다 정확하게 인간 암세포 유래의 이종 3-8보다 종양 이질성을 반영한다. 이 모델은 현재 광범위하게 신약 개발 6,11의 전임상 플랫폼으로 암 생물학 4,12 연구를위한 실험 도구로, 암 치료제 9,10 개인화을위한 플랫폼으로 사용된다. 대부분의 PDXs는 이식 및 실행 가능하게 캘리퍼스를 사용하여 시간이 지남에 따라 종양의 성장을 측정 할 수있는 피하 전파됩니다. 하나전이성 질환은 PDXs를 사용하여 모델링하는 것이 더 어렵다. 특히 유방암, 다른 장기로 전이 용량 이종는 3,5,13 설명되었지만, 전이성 사이트 자발 보급의 빈도는 매우 낮다. 보고 된 경우, 전이성 부담의 식별 및 정량은 사후 표적 기관의 힘든 조직 학적 검사에 의존하고있다. 생물 발광 (루시퍼 라제, 배낭) 또는 형광을 발현하는 암세포는 (녹색 형광 단백질 GFP) 유전자 리포터는 일반적으로 뇌, 폐, 뼈, 간 심내 후 꼬리 정맥 intrafemoral 비장 주입 유방암 전이 실험 모델에서 사용 14-16. 이 모델은 기본 종양에서 보급을 우회하는 동안, 그들은 장기 친 화성 및 전이성 식민지의 메커니즘을 연구 할 가치가있다. 그러나, 기본 환자 종양 및 PDXs에서 파생 된 세포는 낮은 형질 전환 또는 전달 속도 날기를 가질 수있다g 표준 절차. 하나의 대안은 종래의 조직 배양 프로토콜을 사용하여 표시 할 수 체외 (17)에 PDX 유래 세포주를 확립하는 것이다. 이 방법은 그러나, 세포주 유도가 어렵고, 세포의 표현형을 변경할 수있는 가장 PDXs를 라벨에 적합하지 않다. 여기에서 우리는 생체 내 이미징에 적합한 렌티 바이러스 벡터와 PDX-해리 종양 세포의 전달을위한 프로토콜을 제시한다. 또, 면역 쥐에서 해리 루크-GFP 표지 PDX 세포의 심장 내 주사를 사용하여 실험 전이를 설명한다. 리포터 유전자를 발현하는 렌티 바이러스 PDX-해리 organoids의 전달을위한 기본 프로토콜은 이전에 18을 설명 하였다. 현재의 프로토콜에서는 인간 종양 세포에 대한 농축 및 100 % 전달 효율 근처 얻을뿐만 아니라 실험 유방암 검출 용 표지 PDXs 사용하기위한 추가의 방법을 설명전이. 이 프로토콜은 여러 암 다양한 발광 및 형광 마커 PDXs의 종류뿐만 아니라 유전자 발현 (관심의 유전자 즉, shRNA를 최저)의 변조를 라벨에 대해 적용 할 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>BD Biosciences</td>
			<td>354060</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>354001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco/Thermo Fisher</td>
			<td>17504-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungi-antibiotics</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>AM-105-500</td>
			<td>Digestion Buffer</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS Red Ca++/Mg++ free</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex</td>
			<td>Cultrex</td>
			<td>3433-005-01</td>
			<td>Basement Matrix Extract (BME)</td>
		</tr>
		<tr>
			<td>30C shaker</td>
			<td>NewBrunswick Scientific CO. INC</td>
			<td>Series 25 Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>70um filters</td>
			<td>Falcon</td>
			<td>7352350</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpels</td>
			<td>Fisher</td>
			<td>22079690</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine disinfectant</td>
			<td>Durvet&#160;</td>
			<td>NDC# 30798-624-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood&#160; cell lysis reagent</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuraminidase</td>
			<td>Sigma</td>
			<td>N7885-1UN</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (CD326+) microbeads*</td>
			<td>Miltenyil Biotec</td>
			<td>130-061-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lineage cell depletion Kit, mouse*</td>
			<td>Miltenyil Biotec</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator&#160;</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini MACS Magnetic Stand</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>MS or LS columns can be used, adjust to number of cells.</td>
		</tr>
		<tr>
			<td>Illumatool Tunable light system</td>
			<td>Lightools research</td>
			<td>Various</td>
			<td>For in vivo fluorescence imaging</td>
		</tr>
		<tr>
			<td>Xenogen IVIS200 imaging device</td>
			<td>Xenogen</td>
			<td>Various</td>
			<td>For in vivo luminiscence imaging</td>
		</tr>
		<tr>
			<td>Human Cytokeratin Clone MNF116 Monoclonal antibody</td>
			<td>DAKO</td>
			<td>M0821</td>
			<td>Pan-cytokeratin&#160;</td>
		</tr>
		<tr>
			<td>Epidermal Growth factor receptor antibody</td>
			<td>Cell signaling</td>
			<td>4267S</td>
			<td>EGFR</td>
		</tr>
	</tbody>
</table>

labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

이 방법은 높은 역가 렌티 바이러스 벡터 pSIH1-H1-copGFP-T2A-순수하지 않아 및 파지 - EF1aL - 루시 페라 - UBC-GFP-W를 사용하여 PDX-해리 유방암 세포의 전달에 대해 설명합니다. 이 벡터는, 감염 (그림 1a) 후 빠르면 24 시간을 시험관 내에서 전달의 효율성을 추정 허용 형광 마커를 표현한다. 가장 PDXs 들어, GFP 발현 세포 응집체의 형성은 일반적으로 관찰되는이 시점에서, 감염 (도 1b) 후 72 시간까지 지연 될 것이다. 형질 도입은 또는 조에서 PDX-분해 셀 (린 + 세포 고갈,도 1a를 이용하여, IE) 인간 암세포 농축 후에 얻어 질 수있다 (도 1B). 종양의 상피 세포의 보충은 매우 혈관이 종양 마우스 혈액 세포는 생존 세포의 수의 상당한 비율을 나타낸다이었다 권장한다. 체외에서 확인되면 &quot;1&quot;&gt; 표지 된 세포를 수거하고, 세포 외 매트릭스에 현탁하고, NSG 마우스로 재 주입된다. 표지 된 종양 세포를 생물 발광 또는 형광 (도 2)를 이용하여 추적 할 수있는 종양을 재생성. 형질 도입 효율은 다음과 같은 종양 형성을 평가해야하며, 이는 시험 관내 전달 상이한 PDXs의 민감성에 따라 달라진다. 성공적으로 표시된 PDX 100 % GFP 근처 + 1 세대 후 전달 (그림 2b, c) 및 이후의 통로 100 % GFP 근처 +로 유지됩니다 (그림 3)에있을 것입니다. 그러나 일부 PDXs는 체외 전달에 최적를 나타내는, GFP + 세포 (그림 4)의 &quot;누덕 누덕 기운&quot;분포를 보여줍니다. 이러한 경우에, 종양 표지 subpop 확장 재 주입 될 수 GFP + 영역을 선택하는 형광 시청 시스템 아래 해부 될 수있다위험률 또는 종양 분해 될 수 있고, GFP + 세포 NSG 생쥐 재 주입 하였다 FACS를 이용하여 분리 될 수있다. PDX 해리와 전달이 종양 내에서 세포 개체군의 선택을 초래할 수 있기 때문에, 연구자들은 라벨 종양이 밀접하게 파생 된에서 부모의 종양을 닮은 확인해야합니다. PDXs가 중요한 마커와 같은 EGFR, 호르몬 수용체 (즉, 에스트로겐 수용체) 및 팬 cytokeratins을 보여주기 위해 각 세대에 IHC 스테인드한다 (PanCK는, 상피 세포의 마커가) 표시 PDXs에 보존되어있다. EGFR에 대한 염색 5는 그림과 트리플 부정적인 유방암 PDX에서 panCK (이 PDX는 에스트로겐과 프로게스테론 수용체 부족으로 호르몬 수용체 염색이 표시되지 않습니다). 표지 PDXs 자발적인 전이성 확산뿐만 아니라 실험적 전이성을 추적하는데 사용될 수있다순환에 직접 세포를 파종하여 확산. 유방암 PDXs에서 자발적 전이가 자주 발생하지만, 여러 그룹 3,13,25에 의해보고되었다. 전이의 실험 모델은 전이성 캐스케이드 장기 친 화성 및 장기 정착 단계를 연구하는 대안을 제공합니다. 표지 종양에서 해리 세포는 intracardially 주입 및 전이성 부담 생물 발광 영상을 사용하여 추적됩니다. 도 6에 도시 된 바와 같이, PDX가 파지 EF1aL - 루시 페라 UBC-GFP-W 형질 용이 기관 체외에서 생체 내에서 루시퍼 촬상하고 GFP 발현으로 확인 된 폐 및 간에서 250,000 세포 / 마우스 형성된 전이 주입 . <img alt="그림 1" src="/files/ftp_upload/54944/54944fig1.jpg" /> 그림 1 :. 해리 PDX 세포에 형질 도입 효율을 추적 A) </strong&gt;, 상피 세포 농축하지 않고, 별도의 실험에서 세포를 PDX는-해리) 72 시간을 인간 상피 세포 (린 + 고갈) pSIH1-H1-copGFP-T2A-순수하지 않아 렌티 바이러스 입자. B와 전달 후 24 시간 동안 농축 된 세포를 PDX-해리 파지 - EF1aL - 루시 페라 - UBC-GFP-W와 전달 후. 두 패널은 살아있는 세포를 표시합니다. BF : 밝은 필드 이미지는 바는 50 μm의를 나타냅니다. <a href="http://ecsource.jove.com/files/ftp_upload/54944/54944fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54944/54944fig2.jpg" /> 그림 2 :. 설립 호스트 마우스의 레이블이 PDXs 72 시간 동안 파지 - EF1aL - 루시 페라 - UBC-GFP-W와 형질 세포는 여성 NSG 마우스의 유방 지방 패드에 이식했다. 종양은 주입. A) 루시 페라로부터 14 주 동안 성장시켰다<html> 리포터 활성 형광 관찰 시스템을 사용하여 그대로 피부를 통해 관찰 할 수 루시페린. B) GFP 발현의 복강 내 주사 한 후 생체 내 이미징에 의해 평가된다. C) GFP 발현은 종양 표지의 범위를 결정하기 위해 해부에서 검증되어야한다. 이 예는 유비쿼터스 GFP 발현하는 종양을 보여줍니다. <a href="http://ecsource.jove.com/files/ftp_upload/54944/54944fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54944/54944fig3.jpg" /> 그림 3 : 레이블 PDXs는 생체 내에서 여러 구절 후 추적 가능한 마커를 유지 파지 - EF1aL - 루시 페라 - UBC-GFP-W로 표지 유방암 PDX는 전달 후 3 통로를받습니다.. GFP의 발현은 종양 계대에서 거의 100 %로 유지된다.54944 / 54944fig3large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 4" src="/files/ftp_upload/54944/54944fig4.jpg" /> 그림 최적 인 형질 도입 효율과 PDXs 4. 예. A) 유방암 PDX는 차선의 전달이 발생한 후 파지 - EF1aL - 루시 페라 - UBC-GFP-W는 계대 하였다 표지. 그 결과 종양은 GFP +와 GFP- 종양 세포의 혼합 인구를 포함한다. 왼쪽 종양은 더 전파에 적합하지 않습니다. 오른쪽 종양은 형광보고 시스템 하에서 GFP + 영역을 해부에 의해 전파 될 수있다. B) 혼합 종양에서 GFP + 세포의 지역을 쉽게 형광등 아래에서 확인할 수있다. <a href="http://ecsource.jove.com/files/ftp_upload/54944/54944fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 5. PDX의 품질 관리는 통로 (2)에서 트리플 부정적인 유방암 PDX F2-7에서 파라핀 조직에서 EGFR 및 팬 - 사이토 케라틴 (PanCK)의 형질 도입 및과 Passaging. 식 (P2, 전달하기 전에), 종양 후 특징 전달 (P2-I0) 직후에 생성 한 다음 통로 (P2-I1). 20X 이미지는 바는 100 μm의를 나타냅니다. <a href="http://ecsource.jove.com/files/ftp_upload/54944/54944fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 6" src="/files/ftp_upload/54944/54944fig6.jpg" /> 레이블이 PDXs를 사용하여 그림 6. 실험 전이. PDX는 파지 - EF1aL - 루시 페라 - UBC-GFP-W 4에 설명 된대로 3 시간, 및 25 셀 우리를 위해 분해 된 발현하는NSG 마우스의 좌측 심실의 재 주입. A) 전이성 성장은 발광 이미징을 이용하여 살아있는 동물에서 추적 될 수있다. 이 유방암 PDX 전이성 부담 형광 시청 시스템 아래 해부 장기의 생체 외 이미징을 이용하여 B) 간 및 C)의 폐에서 관찰 될 수있다. 바는 약 1cm. 나타낸다 <a href="http://ecsource.jove.com/files/ftp_upload/54944/54944fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

우리는 녹색 형광 단백질과 루시 페라 제 기자를 발현하는 렌티 바이러스 입자 환자 유래의 이종 이식 (PDXs)의 안정적인 표시하는 방법을 설명합니다. 이 방법은 주 사이트 PDXs의 성장을 추적뿐만 아니라, 생체 내 이미징 시스템에서 사용 자발 실험 전이 검출을 허용한다.

종양의 성장과 전이 연구 추적 가능한 기자와 유방암 환자 유래의 이종 이식의 라벨링

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

labeling-breast-cancer-patient-derived-xenografts-with-traceable

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.0K 조회수.  University of Colorado Anschutz Medical Campus.  우리는 녹색 형광 단백질과 루시 페라 제 기자를 발현하는 렌티 바이러스 입자 환자 유래의 이종 이식 (PDXs)의 안정적인 표시하는 방법을 설명합니다. 이 방법은 주 사이트 PDXs의 성장을 추적뿐만 아니라, 생체 내 이미징 시스템에서 사용 자발 실험 전이 검출을 허용한다. 

비디오: Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

종양의 전이에 저산소증의 테스트 효과에 대한 생체 내 모델에서

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

연구

암 연구학

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

육종 환자 유래 이종이식에서 종양 을 발하는 세포의 격리 및 특성화

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

인간 비뇨기과 세포 암 및 대장암 종양 성장 및 자발적 전이를 위한 환자 유래 직교 이종이식 모델

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

하류 분자 분석 및 전이성 전위 평가를 위한 순환 종양 세포의 미세 조작

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Perfused 마이크로 유체 플랫폼에서 환자 유래 제노이식의 Ex vivo 3D 하이드로겔 배양에 대한 향상된 생존력

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

제브라피시의 1차 환자 유래 종양 이종이식의 약물 스크리닝

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

중추신경계 전이의 환자 유래 이종이식 모델의 확립 및 활용

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

제브라피시 애벌레 제노이식 및 종양 행동 분석의 생성

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

환자 유래 유방 오가노이드의 확립 및 배양

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...