저자는 자연 과학 재단의 중국 국가 (31371390), 상태-첨단 개발 프로젝트 (2014AA020702)의 프로그램 및 광 과학 및 기술 (2016B030231001)의 프로그램에서 연구 보조금의 지원을 인정합니다.

모든 동물 작업 동물 복지 행위, 기관 지침에 따라 수행 되었고 기관 동물 관리 및 사용 위원회, 썬 얏-센 대학교에 의해 승인.
1입니다. 준비 편
<ol><li>자 궁 경부 전위에 의해 마우스를 실행 하 고 방언 무 균 수술가 위 핀셋을 사용 하 여 제거.</li>
	<li>3 분, 75% 에탄올에 방언 담가 다음 1.5 mL으로 각 혀를 넣어 10 mM 멸 균 인산 염의 1 mL Eppendorf (EP) 튜브 버퍼링 솔루션 (PBS). 참고: 다음의 모든 단계에서 PBS의 농도이 단계에서 농도와 동일.</li>
	<li>동결 해 동 하 여 세포를 세포: 1 h-80 ° C에서 EP 튜브에 혀를 동결 하 고 다음 3 사이클에 대 일 분 동안 실내 온도에 혀를 녹여.</li>
	<li>수술 봉합 수술 바늘을 사용 하 여의 조각에 각 혀를 로드 하 고 메 마른 은박지의 작은 조각으로 봉합의 끝을 포장. 무 균 조건에서 75% 에탄올을 포함 하는 3.5 cm 또는 6 cm 문화 접시에 작업을 수행 합니다. 참고: 외과 봉합의 각 작품의 적절 한 길이 약 20cm, 그리고 각 조각의 은박지의 적절 한 크기에 대해 0.3 cm2 (1 c m × 0.3 c m). 혀는 은박지 근처 로드 합니다.</li>
	<li>30 미 수행 무 균 조건에서이 작업에 대 한 살 균 PBS의 3 mL와 함께 각 혀 3.5 cm 또는 6 cm 문화 접시에 린스.</li>
	<li>씻어 초순와 방언: 90 µ g/mL의 최종 농도에 불 임 초순의 250 mL 넓은 입 병에 ampicilin를 추가. 방언을 저 어 bar가 포함 된 병에 넣습니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다. 참고: 5 방언까지 놓일 수 있다 봉합의 twining 고려 같은 병에. 석 박 혀 떨어져 미 끄 러에서 방지 하기 위해 병에 봉합의 끝 이다. 혀는 병 안에 남아 있는 봉합의 길이 조정 하 여 2cm 높은 병의 하단에서 배치 되어야 합니다. 이 노트 1.8, 1.10, 1.12, 및 1.16 단계 이기도합니다.</li>
	<li>12 h에 대 한 자력에 병을 넣어.</li>
	<li>NaCl와 방언 세척: 살 균 1의 250 mL 넓은 입 병에 ampicilin를 추가 M NaCl 90 µ g/mL의 최종 농도에. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>트라이 톤 X-100 여 세포 세포: 살 균 2%의 250 mL와 함께 넓은 입 병에 90 µ g/mL의 최종 농도에 ampicilin를 추가 PBS에 트라이 톤 X-100. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>48 h에 대 한 자력에 병을 넣어.</li>
	<li>씻어 CaCl2/MgCl2방언: 90 µ g/mL의 최종 농도에 살 균 5 m m CaCl2/MgCl2 의 250 mL 넓은 입 병에 ampicilin를 추가. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>소화 DNase에 의해: 행 크의의 1 mL 균형 소금물 (HBSS) 각 EP 튜브를 추가. 각각 300의 최종 농도에 DNase HBSS로 추가 µ M.에 각 EP 튜브는 튜브 외부 봉합 부분의 각 혀 이동. 무 균 조건에서이 작업을 수행 합니다. 참고: 이전 단계에서 병 안에 남아 있는 봉합의 일부도이 단계에서 EP 튜브 안에 남아 있는지 확인 하 고 이전 단계에서 병 밖에 남아 있는 봉합의 일부도이 단계에서 EP 튜브 밖에 남아 있다 다는 것을 확인 한다.</li>
	<li>24 h에 대 한 37 ° C에서 EP 튜브에 혀를 품 어.</li>
	<li>씻어 PBS 가진 방언: 90 µ g/mL의 최종 농도에 살 균 PBS의 250 mL 넓은 입 병에 ampicilin를 추가. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>사용까지 준비 편 4 ° C에서 살 균 PBS에 저장 합니다.</li>
</ol>2. TSCC의 3 차원 (3D) 재구성
<ol><li>
정적 TSCC 모델 건설
	<ol>
<li>씨 1.0 x 106 단일 TSCC 셀 (Cal27) 3.5 c m 문화 접시에. Dulbecco의의 3 mL 수정 독수리의 매체/F12 (DF12) 포함 10% 태아 둔감 한 혈 청 (FBS), 90 µ g/mL ampicilin, 및 90 µ g/mL 대 추가 합니다.</li>
		<li>2 ~ 3 일 동안 37 ° C에서 Cal27 셀 문화. 셀 60% 이상 커버 있는지 확인 접시 바닥의 지역.</li>
		<li>문화 접시에 Cal27 단층에 가장을 로드 합니다.</li>
		<li>28 일 동안 37 ° C에서 CO2 인큐베이터에 접시를 넣어.</li>
		<li>셀 문화 과정 매일 문화 매체를 새로 고칩니다. 인큐베이터에서 CO2 농도 5% 이다.</li>
	</ol>
</li>
	<li>
흔들된 TSCC 모델 건설
	<ol>
<li>
성가 냈다 minibioreactor의 준비
		<ol>
<li>10 mL 주사기에서 플런저를 꺼내.</li>
			<li>막대의 낮은 터미널 근처 구멍 (직경 1 ㎝)를 파고 및 구멍에 저 어 바 로드.</li>
			<li>플라스틱 넓은 입 병의 병 뚜껑의 중앙에 구멍 (직경 0.5 c m)를 파고 및 뚜껑을 통해 피스톤 막대를 넣어.</li>
			<li>50 mL 원심 분리기 튜브의 절반을 잘라 고 병 뚜껑의 외부 측면에 용접.</li>
			<li>fishhooks에 묶여있다는 낚시 라인 막대 여 fishhooks 막대를 연결 합니다. 참고: 4 fishhooks까지 장착할 수 막대에.</li>
			<li>압력솥 사용 하기 전에 스스로 만든된 복잡 한입니다. 참고: 플라스틱 넓은 입 병 압력솥 하지를 않습니다. 새로운 살 균 플라스틱 넓은 입 병을 사용 하 여 세포를 배양 하는 동안.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
동적 세포 배양
	<ol>
<li>씨 1.0 x 106 단일 Cal27 셀 성가 minibioreactor에. 10 %FBS, 90 µ g/mL ampicilin, 및 90 µ g/mL 대 포함 된 DF12 매체의 150 mL를 추가 합니다.</li>
		<li>막대에 연결 된 fishhooks를 사용 하 여 minibioreactor에 가장을 로드 합니다.</li>
		<li>병 뚜껑을 강화 하 고는 자력에 minibioreactor를 넣어. 7 ~ 14 일 동안 37 ° C에서 CO2 배양 기에서 200 rpm에서 minibioreactor를 활성화 합니다. 참고: CO2 배양 기에서 CO2 의 농도 5%.</li>
	</ol>
</li>
</ol>

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저자는 공개 없다.

Decellularized ECM 제조에 잘 설립 프로토콜 조직에서 세포 구성 요소를 제거 하는 동안 네이티브 ECM 구성 유지 한다 거의 완전 하 게21. 현재 보고 된 decellularization 프로토콜 대류 수송에 의해 세포 물질을 제거 하는 맥 관 구조를 통해 관류를 요구 하는에 불구 하 고 기계적인 동요 채택 되었다 여기, 전통적인 간단 하 고 저렴 한 방법22 로 알려진 , <sup class="x...

혀는 deglutition, 조음, 시음 등 다양 한 중요 한 기능이 있다. 따라서, 혀 기능 장애 환자 들의 삶의 질1에 큰 영향을가지고. 구강에서 가장 일반적인 악성 종양은 혀 편평 세포 암 종 (TSCC), 일반적으로 알코올 음료 또는 담배2를 연기 하는 사람에서 생기는.
최근 몇 년 동안, 작은 진행 TSCC에 기초 연구에 달성 되었습니다. 효율적인 생체 외에서 연구의 부족 모델 가장 큰 문제 중 하나 남아 있다. 따라서, 기질 (ECM) 잠재적인 솔루션으로 밖으로 변합니다. ECM 고도로 조직된 매트릭스 구성 요소 구성 하는 복잡 한 네트워크 프레임 이기 때문에, 비 계 재료는 ECM 같은 구조와 구성 암 연구에 대 한 능력이 될 것 이다. Decellularized ECM는 같은 기원에 생체 외에서ECM의 가장 중요 한 이점을 밝혀에서 셀에 대 한 완벽 하 게 틈새를 제공할 수 있습니다.
ECM 세제와 효소를 사용 하 여 decellularization 통해 조직에서 제거 되는 세포 구성 성분으로 유지 될 수 있습니다. Decellularized 매트릭스의 콜라겐, fibronectin, 및 laminin를 포함 하 여 다양 한 ECM 구성 요소, 배양된 세포 생존, 확산, 그리고 셀3의 홍보에 대 한 기본-조직-같은 microenvironment을 제공 합니다. 또한,이 식 위한 immunogenicity ECM에 세포 구성 성분의 부재와 함께 최소한의 수준으로 줄일 수 있습니다.
지금까지, decellularized ECM에 대 한 제작 방법은 다른 조직 및 장기 심장4,5,,67년 간8,9,10 등에서 시도 되었습니다. ,11, 폐12,13,14,15,,1617및 신장18,19 , 그러나 20., 관련 연구 우리의 지식 최선 다 해 혀에서 비슷한 작품에서 찾을 수 있다.
이 연구에서는 decellularized 혀 기질 (가장) 물리, 화학, 및 효소 치료의 일련에 의해 효율적으로 그리고 싸게 조작 했다. 다음은 가장 TSCC 동작 및 개발에 대 한 적절 한 시뮬레이션을 보여주는 TSCC에서 생체 외에서정리에 사용 되었다. 가장 좋은 생체 적합성으로 가장 TSCC 연구3큰 잠재력을 가질 수 있음을 나타내는 조직-특정 틈새에 셀 가이드 수 있다. 여기에 표시 된 프로토콜 연구자 중 병 또는 TSCC의 임상 치료에 대 한 선택을 제공 합니다.

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			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
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			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
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			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
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			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
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			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
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			<td style="width:119px;">153066</td>
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			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
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			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
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			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
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			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
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			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
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			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
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			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
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			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
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			<td style="width:119px;">227270</td>
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			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
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			<td>Tongue squamous cell carcinoma cell line</td>
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			<td>Human osteosarcoma cell line</td>
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			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
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			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
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			<td height="21" style="height:21px;width:216px;">Water purifier</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:292px;">BLAU</td>
			<td style="width:119px;">717805</td>
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fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

혀 편평 세포 암 (TSCC) 시험관에대 한 효과적이 고 현실적인 모델을 생성 하기 위해 방법은 생산 decellularized 혀 기질 (가장) TSCC 건설 기능 건설 기계를 제공 하 게 만들어졌습니다. 가장 세포 성장, 감 별 법 및 셀 마이그레이션에 대 한 생체 외에서 틈새를 제공합니다. 원시 세포 외 기질 (ECM) 및 decellularized 매트릭스에 유지 하는 생 화 확 적인 작곡의 마이크로 구조 정박 하는 셀에 대 한 조직의 특정 틈새를 제공 합니다. 가장의 제조 deoxyribonuclease (DNase) 소화 유기 또는 무기 전처리의 심각한 동반에 의해 실현 될 수 있다. 이 프로토콜은 운영 하 게 쉬운 하 고는 decellularization에 대 한 높은 효율을 보장. 가장 TSCC 모델의 건설을 가능 하 게 TSCC 셀 정적 또는 흔들 문화 조건에 대 한 유리한 cytocompatibility를 보여주었다. 스스로 만든된 생물 세포 배양에 대 한 영구 촉발된 조건 또한 사용 되었다. 가장을 사용 하 여 복원된 TSCC 보여주었다 임상 TSCC histopathology, 닮은 속성과 특성 TSCC 연구 잠재력을 제안.

이 프로토콜의 가장 준비에 대 한 효율적이 고 적절 한 것으로 판명. 가장 완벽 한 decellularization 국어 조직 비교 했다. Decellularization의 효능 hematoxylin-오신 (그) (그림 1A-B) 얼룩에 의해 확인 되었다. 그 결과 얼룩 가장 (그림 1B)에서 핵 얼룩의 완전 한 실종을 밝혔다. 또한, 이전 작품에서 DNA 콘텐츠 정량화?...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

혀 기질과 혀 편평 세포 암 종에서 생체 외에서 의 재구성의 제조

메서드는 효율적인 decellularization와 혀 세포 외 매트릭스 (가장)의 준비에 대 한 여기 표시 됩니다. 가장 정적 또는 흔들 문화 조건 하에서 혀 편평 세포 암 종 (TSCC) 모델의 재구성 기능 건설 기계로 사용할 수 있습니다.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

6.2K 조회수.  Sun Yat-sen University. 메서드는 효율적인 decellularization와 혀 세포 외 매트릭스 (가장)의 준비에 대 한 여기 표시 됩니다. 가장 정적 또는 흔들 문화 조건 하에서 혀 편평 세포 암 종 (TSCC) 모델의 재구성 기능 건설 기계로 사용할 수 있습니다.

비디오: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

머리와 목 편평 ​​세포 암에서 신경초 침략을위한 모델

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

연구

암 연구학

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

전임상 치료의 양적 및 종 방향 평가를위한 동계 마우스 모델 전이성의 신장 세포 암

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

포르말린 - 고정, 파라핀 - 임베디드 인간 구강 성 편평 세포 암종 샘플에서의 T 세포 아군의 4 색 형광 면역 조직 화학

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

머리와 목 편평 상피 세포 암에 대 한 회전 타원 체 기반 3D 셀 문화 모델에서 테스트 하는 치료

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

종양 면역 프로 파일링에 대 한 쥐 및 치료 반응 평가에 편평 세포 암 종 세포의 intramucosal 접종

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

다리에 대 한 3 차원 뼈 세포 외 매트릭스 모델

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

세포외 매트릭스 하이드로겔을 사용하여 정상 조직 방사선 효과 연구

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1 대식세포-4T1 마우스 유방 암암 세포에서 식세포 활동의 타임랩스 2D 이미징

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

마우스 신장과 닭 융모알란투암 막에 설립된 전이성 맑은 세포 신장 세포 암종 모델 비교

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

폐 편평상피암 진행을 모델링하는 다차원 동문화 시스템

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...