우리 기술 및 사무 작업에 대 한 양 제이 시노하라와 I. Nieda 감사합니다. 또한, 우리 감사 박사 시로 마 츠 모토, 則 Haruta, 켄 타로 쿠라 시, 그리고 카즈야 타카하시 수술 실에 샘플 수집에 대 한 그들의 협력에 대 한. 이 작품은 특정 교육 과학기술부, 과학, 스포츠, 그리고 일본 문화 및 일본 사회에서 과학적인 연구를 위한 과학 진흥 (17 K 10606)에 대 한 지원 했다.

LDN는 환자가이 연구에 등록에서 가져온 하 고 기관 검토 위원회의: Jichi 의학 대학에 의해 승인 했다.
1. 복 부 구멍 Lavages 및 NET 검출에서 LDN의 격리
<ol><li>샘플 수집<ol>
<li>위장 암 복 부 수술을 받은 환자에 상처 폐쇄 하기 전에 복 부 구멍에 직접 일반적인 살 균 염 분의 1000 mL를 달 이다. 참고: 샘플은 연령이 나 성별에 따라 바이어스 없이 위절제술, colectomy, 또는 esophagectomy를 수술 하는 환자에서 얻은 했다. 식 염 수는 컨테이너로 전송 되었고 1 분 이내 전체 복 부에 부 어. 이것은 환자에 중요 한 영향 없이 수술 후 복 막 세척으로 정기적으로 수행 됩니다.</li>
		<li>게 1 분 이상 동안 광범위 하 게 복 부 구멍. 참고: 예제는 균일 한 외과 의사의 손에 생긴된 액체 흔들 천천히 하는 것이 좋습니다.</li>
		<li>4 50 mL 주사기와 게의 200 mL를 복구 합니다. 참고: 때로는 고무 커넥터 체액을 데 사용 됩니다.</li>
	</ol>
</li>
	<li>Granulocyte 특정 mAb, CD66b16를 사용 하 여 복 막 LDN의 정화를 수행 합니다. 참고: 많은 단 세포 조밀도 기온 변화도 원심 분리 후 중간 계층에 포함 된 때문에 긍정적인 선택의 변이 호 중구 CD66b mAb를 사용 하 여 수행 되었다.<ol>
<li>50 mL 튜브에 복 막 게 유체를 전송. 참고:이 단계에서 불순물을 제거 하는 100 µ m 나일론 필터를 통해 액체를 전달 합니다.</li>
		<li>실시간에서 7 분 동안 270 x g에서 복 막 액체 원심</li>
		<li>0.02 %EDTA PBS의 5 mL에 산 탄을 resuspend.</li>
		<li>조심 스럽게 3 mL 밀도 그라데이션 솔루션에 세포 현 탁 액의 5 mL을 오버레이.</li>
		<li>휴식 없이 RT에서 15 분 동안 1700 x g에서 원심.</li>
		<li>피 펫을 사용 하 여 중간 레이어 (그림 1A)를 포함 하는 솔루션의 2 ~ 3 mL를 수확 하 고 0.02 %EDTA PBS의 10 mL와 함께 그것을 혼합.</li>
		<li>RT에 7 분에 대 한 400 x g에서 복 막 액체 원심 및 삭제는 상쾌한.</li>
		<li>실시간 삭제는 상쾌한에 7 분 동안 270 x g에서 원심 분리기와 0.02 %EDTA, PBS의 또 다른 10 mL를 추가 합니다.</li>
		<li>마그네틱 셀 분리 키트에 대 한 버퍼의 60 µ L에 셀 펠 릿 (1 x 107)를 용 해.</li>
		<li>펠 릿을 Fc 블록의 20 µ L을 추가 하 고 4 ° c.에서 10 분 동안 품 어</li>
		<li>20 µ L 안티-CD66b의 microbeads와 활용 및 4 ° c.에 추가 10 분 동안 품 어 추가</li>
		<li>세척 하 고 다시 맥 버퍼의 500 µ L에서 알 약을 중단.</li>
		<li>적당 한 자석 분리기의 자기장에서 자석 열에 세포 현 탁 액을 적용 하 고 버퍼의 15 mL과 열 세척 하 여 흐름을 통해 포함 된 CD66b(-) 세포를 수집 합니다.</li>
		<li>마그네틱 구분 기호에서 열을 제거 하 고 즉시 단단히 열에 플런저를 밀어 여 자석으로 레이블이 지정 된 CD66b(+) 세포 밖으로 플러시. 참고: CD66b(+) 세포 인구 구성 되어 주로 호 중구 나타내는 FACS 분석에 의해 결정으로 > CD66(+) 분수의 95%는 CD11b, CD15, 및 CD16, 긍정적인 CD14에 대 한 부정적인.</li>
	</ol>
</li>
	<li>그물의 세대<ol>
<li>RT에서 7 분 동안 270 x g에서 원심 분리 후 다시 격리 LDN를 일시 중단 (5 x 106) 10 %FCS RPMI1640의 1 ml에서.</li>
		<li>5% CO237 ˚C에서 2 h 6-잘 폴 리-L-리 신 코팅된 접시 (직경에서 6 cm)에 LDN 문화.</li>
		<li>5 µ M의 최종 농도에서 녹색 형광 counterstain (핵과 염색체 얼룩 막 스며들 지 않는 염료)를 추가 합니다.</li>
		<li>즉시 그물 (extracellular DNA 구성 요소에서 형광 현미경 검사 법에서 LDN 추방)의 형태를 관찰 합니다.</li>
	</ol>
</li>
</ol>2. 붉은 형광 셀 링커 염료와 종양 세포의 얼룩
<ol><li>인간의 암 세포 라인 MKN45, NUGC, 및 OCUM-1 준비.</li>
	<li>1 x 107 셀 PBS + 0.02 %EDTA 15 mL와 실시간에 7 분 동안 270 x g에서 원심 분리기 세척</li>
	<li>펠 릿, 얼룩 염료에 대 한 솔루션의 1 mL을 추가 하 고 부드럽게 플라스틱.</li>
	<li>얼룩에 대 한 솔루션의 1 mL에 빨간색 형광 셀 링커 염료의 4 µ L를 녹이 고 세포 현 탁 액 단계 2.2에서에서 혼합.</li>
	<li>실시간에서 4 분 동안 쥐 똥을 품 어</li>
	<li>DMEM의 4 개 mL를 추가 (10 %FBS) 착 색 반응을 중지 하.</li>
	<li>270 x g 7 분 원심 하 고 삭제는 상쾌한.</li>
	<li>2.6-2.7 단계를 두 번 반복 합니다.</li>
	<li>형광 현미경으로 확인 (여기 551 = nm, 방출 = 567 nm) 종양 세포 붉은 얼룩이 있습니다.</li>
</ol>3. 그물에 종양 세포 접착의 분석
<ol><li>다시 0.1 %BSA 보충 RPMI 1640의 1 mL에 빨간색 형광 스테인드 종양 세포 (1 x 106) 일시 중단.</li>
	<li>1.3 단계에서 설명한 대로 그물을 생산 LDN 문화에 종양 세포를 추가 합니다.</li>
	<li>종양 세포는 그물에 연결할 수 있는 37 ˚C에서 5 분 동안 품 어. 참고: 긴 외피 종양 세포 접착 LDN 하거나 번호판을 유도합니다.</li>
	<li>매체를 제거 하 고 부드럽게 prewarmed 미디어 (0.1 %BSA + RPMI 1640)의 2 개 mL를 추가 하 고 접시를 소용돌이 여 우물을 씻어.</li>
	<li>3.4 단계에서 세척 절차를 두 번 반복 합니다. 참고: 그물부터 약하게 플레이트에 부착, 세척 부드럽게 스스로 그물의 제거를 피하기 위해 가능한 이루어져야 합니다.</li>
	<li>핵 및 그물의 시각화를 위한 5 µ g/mL의 최종 농도에 염색체 얼룩이 지기에 대 한 녹색 형광 염료를 추가 합니다.</li>
	<li>그물을 관찰 하 고 적절 한 필터를 사용 하 여 종양 세포를 첨부 (녹색, 여기 = 504 nm, 방출 523 = nm; 레드, 여기 551 = nm, 방출 = 567 nm).</li>
	<li>종양 세포는 그물에 의해 갇혀 보여 인물을 병합 합니다. 참고: 일부 실험에서 DNA 분해 효소에 추가 되었습니다 LDN 문화 (최종 농도 100 U/ml) 5 분에 대 한 공동 부 화 하기 전에 5 분.</li>
</ol>4. 시간 경과 비디오 분석 갇혀 종양 세포의
<ol><li>문화 35 m m에서 복 LDN 라운드 접시 코팅 폴 리-L-리 신 1.3 단계에서 설명한 대로.</li>
	<li>핵 및 1.3 단계에서 설명한 대로 그물을 시각화 하는 염색체 얼룩이 지기에 대 한 녹색 형광 염료를 추가 합니다.</li>
	<li>외피의 1 분 후 미디어를 제거 하 고 0.1 %BSA + RPMI 1640의 2 개 mL를 추가 합니다.</li>
	<li>미디어를 제거 하 고 흠 없는 1 x 106 MKN45 세포 정지 0.1 %BSA + RPMI 1640 추가. 실시간에서 5 분 동안 품 어</li>
	<li>3.4 단계에서 설명한 대로 부드럽게 세척 하 여 모든 첨부 되지 않은 종양 세포를 제거 하 고 10% fcs 100 u/mL 페니실린와 100 µ g/mL 스 보충 DMEM 추가.</li>
	<li>문화 요리 전체 보기 셀 관측 시스템에 탑재 하 고는 종양 세포는 그물에 의해 갇혀 있다. 적절 한 필드를 선택 합니다.</li>
	<li>계속 추가 대 한 공동 2 일 문화 하 고 정상적인 빛과 형광, MKN45 세포와 그물을 각각 감지에서 6 분 마다 필드의 연속 사진을 찍어.</li>
	<li>각 timepoint에 이미지를 첨가 하 고 시간 경과 비디오 이미지 뷰어 소프트웨어를 사용 하 여 구성.</li>
</ol>

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저자 들은 아무 경쟁 금융 관심사 선언 합니다.

이전 연구는 종양 세포를 순환 수 있습니다 갇혀 있을 NET 기판10, vivo에서11보고 있다. 전이성 유방암 세포 자극 하는 호 중구 및 대상 기관17에서 종양 세포 성장에는 그물의 대형을 유도 표시 되었습니다. 또한, 우리는 수술 후 게 액체에서 LDN의 단기 문화 수 효율적으로 더 자극15없이 종양 세포를 모 함 하는 거...

혈액 순환에 다형 핵 호 중구 밀도 그라데이션 준비 방법을 통해 단 세포에서 일반적으로 분리 된다. 그러나, CD11b(+), CD15(+), CD16(+), 그리고 CD14(-) 고기와 저밀도 neutrophils (LDN)로 알려진 일부 호 중구는 공동 단 세포로 순화. LDN의 상대 수 면역 질환1,2, 암4,5, 패 혈 증3, 등 다양 한 병 적인 조건에서 크게 증가 합니다. 이전 연구는 LDN의 호 중구6phenotypically 및 기능적으로 뚜렷한 클래스 나타났습니다. 그것은 혈액 순환에 LDN 더 정상적인 밀도 호 중구2,7보다 호 중구 extracellular 함정 (그물)를 생산할 가능성이 있습니다 주목 해야한다. 그물 핵 산, 히스톤, 프로 테아 제, 그리고 세밀 하 고 cytosolic 단백질의 구성 하는 웹-같은 구조 이며, 그들은 효율적으로 모 함 하 고 병원 균8파괴.
최근, 그물 캡처 뿐만 아니라 미생물, 하지만 또한 혈소판 혈전 형성9 및 종양 전이10,11에 지원할 수 있는 종양 세포를 순환 표시 되었습니다. 그러나, 뒤에 그물과 혈소판 또는 종양 세포 사이 접착 상호 작용 분자 메커니즘 여전히 명확 하지 않다. 더 최근에, 생체 외에서 접착 분석 결과 골수성 백혈병 세포 (K56212) 폐 암 세포 (A54913) 연결 및 그물을 β1와 β3 integrins를 통해 밝혔다. 저자 호 중구에서 고립 그리고 phorbol myristate 12 13-아세테이트 (PMA) 접착 기판14에 의해 활성화 인터넷 주식 사용. 이 분석 결과 호 중구의 부재에서 NET 구성 요소와 실제 상호 작용의 탐지를 허용, 그것은 arguable 여부 그물 생산 에서 동일한 분자 구조를 유지는 "셀-무료 인터넷 주식" 고속 원심 분리에 의해 분리 vivo. 최근에, 우리는 복 부 수술 포함 거 대 한 그물을 생성 하 고 복 막 전이15발생 하는 종양 세포에 연결 된 많은 성숙한 LDN 후 그 게 복 막 액체를 발견. 이 연구에서 우리는 성공적으로 어떤 물리적 조작 없이 그대로 그물에 종양 세포의 접착을 시험 했다. 여기, 우리는 그물과 무료 종양 세포 사이 접착 상호 작용을 검출 하는 기법의 세부 정보를 보여줍니다.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare, SWEDEN</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>StraightFrom&#8482; Whole Blood CD66b MicroBeads</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-104-913</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Rinsing Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Magnetic Separator</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-042-501</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX green nucleic acid stain 5mM solution in DMSO</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9691</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium-high glucose (DMEM)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5mol/l-EDTA Solution (pH 8.0)</td>
			<td>nacalai tesque, Japan</td>
			<td>06894-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>gibco by life technologies, Mexico</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin lyophilized powder, &#8805;96% (agarose gel electrophoresis)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies Japan</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin Prophylactic</td>
			<td>InvivoGen, San Diego, CA-USA</td>
			<td>ant-mpp</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington, Lakewood NJ)</td>
			<td>LS002138</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 6Well</td>
			<td>IWAKI, Japan</td>
			<td>4810-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 24Well</td>
			<td>IWAKI, Japan</td>
			<td>4820-040</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein stereomicroscope</td>
			<td>BX8000, Keyence, Osaka Japan</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole view cell observation system</td>
			<td>Nikon, Kanagawa, Japan</td>
			<td>BioStudio (BS-M10)</td>
			<td></td>
		</tr>
		<tr>
			<td>MKN45 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NUGC-4 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OCUM-1 human gastric cancer cell line</td>
			<td>Osaka City University, Japan</td>
			<td>N/A</td>
			<td>Gift from Dr. M.Yashiro</td>
		</tr>
	</tbody>
</table>

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

호 중구 릴리스 호 중구 extracellular 함정 (그물), 캡처 및 미생물을 파괴할 수 있는 활성화. 최근 연구 제안 그물 자가 면역 질환, 혈전 증, 및 종양 전이 등 다양 한 질병 과정에서 포함 된다. 여기, 우리는 그물을 부착 후 성장 무료 종양 세포의 트래핑 동안 NET 활동을 감지 하 상세한 생체 외에서 기술을 보여줍니다. 첫째, 우리는 laparotomies 수술 환자에서 수술 후 복 막 게 액체에서 낮은 밀도 neutrophils (LDN)를 수집. LDN의 단기 경작 녹색 형광 핵과 염색체 counterstain 가시화 했다 거 대 한 그물 형성 결과. 인간의 위 암 세포 라인 MKN45, OCUM-1 및 NUGC-4 그물의 공동 화, 후 많은 종양 세포는 그물에 의해 갇혀 있었다. 그 후, 첨부 파일 DNase I. 계시 노출 인 비디오 공개 종양 세포는 그물에 의해 갇혀 죽지 않 았와 그물의 저하에 의해 완전 하 게 폐지 했다 하지만 대신 적극적으로 지속적인 문화에서 성장 했다. 이러한 메서드는 그물과 다양 한 유형의 세포 및 물자 사이 접착 상호 작용의 탐지에 적용할 수 있습니다.

2 시간 문화에서 CD66b(+) LDN 복 막 게 유체 보였다 문자열 구조에서 파생 된에 대 한 녹색 형광 염료와 스테인드 핵 및 단 셀 하지 않았다 CD66b(-) 동안 염색체 (그림 1B), (그림 1C). 그러나 때 LDN 문화 했다 청소용으로 100 U/mL DNase 나, 특징적인 구조는 파괴 (그림 1D), 그들은 호 중구에서 추방 하는 extracellular DNA의...

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

여기, 우리는 인간의 저밀도 호 중구 (LDN), 수술 후 복 막 게 액체에서 회복 대규모 호 중구 extracellular 함정 (그물)를 생산 하 고 이후에 성장 하는 무료 종양 세포를 효율적으로 트랩 방법을 제시.

호 중구 Extracellular 함정 밀도가 낮은 호 중구에 의해 생성 된 종양 세포 성장 및 첨부 파일 중재 하십시오 복 막 게 액체에서 얻은

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

neutrophil-extracellular-traps-generated-low-density-neutrophils

Research

JoVE Journal

Cancer Research

8.7K 조회수.  Jichi Medical University. 여기, 우리는 인간의 저밀도 호 중구 (LDN), 수술 후 복 막 게 액체에서 회복 대규모 호 중구 extracellular 함정 (그물)를 생산 하 고 이후에 성장 하는 무료 종양 세포를 효율적으로 트랩 방법을 제시.

비디오: Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

폐 종양 세포 모집 분석 결과

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

연구

암 연구학

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

단일 셀 다운스트림 특성화에 대 한 대상 셀 사전 농축 및 전체 게놈 증폭

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

GFP Lentivirus 불리고 Organoids 환자 파생 결 장 종양에서 경작에 의해 생성 된 Micrometastases의 고감도 검출

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

라이브 셀 이미징 분석 실험 연구 세포종 뇌 종양 줄기 세포 이동과 내 습을

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

조작된 종양 관련 호중구를 종양 베어링 마우스로 옮겨 생체 내 혈관신생 잠재력을 연구합니다.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

면역형광에 의한 비소세포 폐암 환자의 순환 종양 세포의 반자동 PD-L1 특성화 및 열거

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

종양 성장에서 암 관련 섬유아세포의 역할을 조사하기 위한 마우스 모델

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

낮은 종양 함량 조직에서 종양 농축을 위한 자동화된 해부 프로토콜

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.