우리 Dov Zipori (바이 츠만 과학 연구소, Rehovot, 이스라엘) 친절 하 게 제공에 감사 우리 murine 골 수 기질 세포 라인 14F1.1. 이 연구는 중국 국가 자연과학 기초 (81771729 번)과 충칭 의료 Yongchuan 병원 대학 (개에서 교부 금에 의해 지원 되었다 YJQN201330; YJZQN201527)입니다.

참고:이 프로토콜에서 사용 하는 모든 셀 냉동된 재고 셀에서 녹고 후 적어도 3 세대에 대 한 성장 해야 합니다.
1. 수확 흑색 종 세포 파생 요소
<ol><li>
준비
	<ol>
<li>B16F10 세포 및 마우스 흑색 종 세포 라인을 얻을. 참고:이 프로토콜에 대 한 마우스 흑색 종 세포 선 과학의 중국 아카데미의 줄기 세포 은행에서 얻은 했다.</li>
		<li>B16F10 세포 배양 (100 mL)에 대 한 완벽 한 매체를 확인 합니다. Dulbecco의 수정이 글의 중간 (DMEM) 10% 태아 둔감 한 혈 청 (FBS), 페니실린, 50 U/mL 및 50 µ g/mL 스 보충을 사용 합니다.</li>
		<li>혈 청 무료 매체 B16F10 세포 (50 mL)의 기아에 대 한 확인 합니다. DMEM 매체 FBS 없이, 페니실린, 50 U/mL 및 50 µ g/mL 스 사용 합니다.</li>
	</ol>
</li>
	<li>
흑색 종 조절 매체 (CM)를 준비
	<ol>
<li>100 m m에서 씨 2.0 x 105 B16F10 세포 10%의 10 mL와 FBS DMEM 요리. 다음 24 h에 대 한 5% CO2 의 분위기 속에서 37 ° C 배양 기에서 세포를 품 어.</li>
		<li>셀 합류의 약 80%에 도달, 문화 매체를 제거, 인산 염 버퍼 식 염 수 (PBS) (1x, pH 7.4)의 5 mL로 두 번 셀을 세척 하 고 10 mL 혈 청 무료 매체는 기아에 대 한 매체를 바꿉니다.</li>
		<li>18-24 h에 대 한 보육을 셀을 반환 합니다.</li>
		<li>기아, 후 한 피 펫과 바른된 매체 (CM)를 수집 하 고 신선한 50 mL 원심 분리기 튜브에 그것을 전송. 참고: 조절된 매체 B16F10 세포 배양의 상쾌한입니다. B16F10 세포 CM를 수집 하기 전에 좋은 조건에는 다는 것을 확인 하십시오.</li>
		<li>1000 x g 4 ° c.에 5 분에 대 한 CM을 원심</li>
		<li>플라스틱은 상쾌한 하 고 신선한 무 균 튜브 또는 병에 그것을 전송. 참고: 아래쪽 튜브의 펠 릿에서 pipetting 하지 마십시오.</li>
		<li>셀 파편 제거, 살 균 병 또는 튜브 주사기 연결 0.45 μ m 필터를 통해 그것을 전달 하 여 CM을 필터링. 참고: 0.22 μ m 필터는 CM을 필터링 하는 데 적합 합니다.</li>
		<li>-20 ° C에서 필터링 된 상쾌한 사용까지 저장 합니다. 참고: 그것은-20 ° C에서 상쾌한 1 달 이상에 대 한 저장 하지 것이 좋습니다. 또는, 체형의 분화에 대 한 시간 더 연장된 기간에 대 한-80 ° C에서 CM을 저장 합니다.</li>
	</ol>
</li>
</ol>2. 흑색 종 세포에서 파생 된 요소에 의해 골 Adipocytes의 유도
<ol><li>
준비
	<ol>
<li>Adipogenic 유도 중간28의 100 mL를 확인 합니다. 완전 한 DMEM 인슐린, 0.5 m m 3-isobutyl-1-methylxanthine, 1 µ M dexamethasone의 5 µ g/mL와 보완을 사용 합니다. 4 ° c.에 2 주까지 저장하시오</li>
		<li>Adipogenic 유지 보수 매체의 100 mL를 준비 합니다. 완전 한 DMEM 사용 또는 50%의 흑색 종 5 µ g/mL의 인슐린으로 보충 하는 CM와 혼합.</li>
		<li>기름 빨간 O 재고 솔루션을 확인 합니다. 소 프로 파 놀 (≥ 99.5%)의 10 mL에 기름 빨간 O 가루 30 mg을 녹이 고 소용돌이 의해 잘 그들을 믹스. 참고: 4 ° c.에서 1 년 저장</li>
		<li>기름 빨간 O 작업 솔루션을 확인 합니다. 증기 두건에서 3: 2의 비율에 있는 이온된 수로 (2.1.3 단계)에서 기름 빨간 O 재고 솔루션을 희석. 실 온에서 15 분 동안 혼합물을 품 어 하 고 사용 하기 전에 0.22 μ m 필터를 통해 필터링. 참고:만 2 시간 안에 신선한 작업 솔루션을 사용 합니다.</li>
	</ol>
</li>
	<li>
체형 차별화
	<ol>
<li>문화 stromal 세포 5% CO2의 분위기 속에서 37 ° C 배양 기에서 80 %confluency 완전 한 DMEM와 60 mm 접시에 마우스 골 수29 에서 14F1.1 라인. 참고:은 주 골 수 유래 중간 엽 세포3014F1.1를 또는 바꿉니다.</li>
		<li>1-2 분 동안 인큐베이터에 0.25 %trypsin 솔루션의 1.5 mL와 셀 trypsinize</li>
		<li>셀에 10 %FBS 매체의 5 mL을 추가 하 고 유리 hemocytometer;와 셀 숫자의 개수 계산 다음 1-2 105/mL x 약 셀 밀도 조정 합니다.</li>
		<li>씨앗 5 x 104 24-잘 접시에 잘 당 14F1.1 세포의 adipogenic 유도 매체의 0.5 mL와 함께. 2 일 후 부 화, 셀 합류의 약 90%에 도달. 참고:이 단계는 매우 중요 한입니다. 골 수 기질 세포 adipogenic 유도 매체 없이 adipocytes에 유도 될 수 없습니다.</li>
		<li>유도 매체를 제거 하 고 씻어 1 mL의 PBS로 두 번 셀 때마다.</li>
		<li>Adipogenic 유지 보수 매체의 1 mL를 변경 하 고 adipocyte 성숙까지 셀 문화 계속. 참고: 현미경, adipocytes 소형 또는 대형 비누 방울 처럼 모양과 아주 쉽게 인식할 수 있습니다. 일반적으로, 성숙 adipocytes 6번째 또는 8번째 날에는.</li>
		<li>기름 빨간 O와 성숙한 adipocytes 얼룩 또는 adipocytes 정량적 중 합 효소 연쇄 반응 (정량) 분석을 위해 수집.</li>
	</ol>
</li>
	<li>
체형 얼룩
	<ol>
<li>워시 차별화 된 14F1.1 세포를 PBS로 2 배 및 1 h 3.7% 포름알데히드에 그들을 수정.</li>
		<li>60% 소 프로 파 놀 셀 린스를 완전히 건조 하도록 허용 합니다.</li>
		<li>우물에 기름 빨간 O 작업 솔루션의 0.2 mL를 추가 하 고 0.5 ~ 2 h에 대 한 실 온에서 품 어.</li>
		<li>셀 언바운드 염료를 제거 하는 물으로 씻는 다.</li>
		<li>가벼운 현미경 염색된 adipocytes 사진.</li>
	</ol>
</li>
</ol>3. 흑색 종 세포와 골 Adipocytes의 coculture
<ol><li>
준비
	<ol>
<li>성숙한 뼈 골 수 adipocytes 준비: 2 일, adipogenic 유도 매체와 24-잘 접시에 성숙한 BM adipocytes에 14F1.1 세포를 유발 하 고 다음 추가 6-8 일에 대 한 adipogenic 유지 보수 매체에서 그들을 유지.</li>
		<li>B16F10 세포 준비: DMEM 매체의 5 mL와 60mm 요리에서 B16F10 세포 문화.</li>
		<li>축 축 하 게 막 삽입 (예: transwell): 0.4 µ m 기 공 크기 막 삽입을 FBS 무료 DMEM 매체의 150 µ L을 추가 하 고 24-잘 접시 가득 FBS 무료 DMEM 매체의 500 µ L의 우물에 그들을 담가.</li>
	</ol>
</li>
	<li>
Coculture 설정
	<ol>
<li>천천히 각 삽입의 매체에서 피 펫입니다. 주: 매체를 분리할 때 막을 삭제 하지 마십시오.</li>
		<li>3.2.1 단계에서 시드 B16F10 세포 (0/103/105/106) 0.4 µ m 기 공에 삽입합니다.</li>
		<li>3.1.1 단계에서 성숙 골 adipocytes에 우물에 adipogenic 유지 보수 매체를 바꿉니다. 완전 한 DMEM 매체의 600 µ L와.</li>
		<li>Coculture 실험 (3.2.3 단계)에서 성숙 골 adipocytes와 우물에 (에서 단계 3.2.2) B16F10 세포로 가득 삽입 전송.</li>
		<li>2 일 동안 37 ° C, 5% CO2 cocultures를 품 어.</li>
		<li>외피의 48 h 후는 coculture의 삽입을 제거 하 고 다음 씻어 adipocytes 2 x 1 x PBS의 500 µ L. 참고: 필요한 경우 필요에 따라 분석에 대 한 삽입에서 B16F10 세포를 수집 합니다.</li>
		<li>기름 빨간 O 작업 솔루션 adipocytes 얼룩 또는 정량 분석을 위한 adipocytes 수집. 주: 단계 2.3에서에서 착 절차를 참조 하십시오.</li>
		<li>가벼운 현미경 염색된 adipocytes 사진.</li>
	</ol>
</li>
</ol>4. 정량 분석 체형 관련 유전자 서명의
<ol><li>24-잘 접시에서의 준비에 사용 RNA 격리 시 약 잘 당 0.5 mL를 추가 성숙한 체형에 검색할 준비가 되어.</li>
	<li>RNA 추출 및 cDNA 합성31에 대 한 표준 프로토콜을 따릅니다.</li>
	<li>18S와 GAPDH 뇌관을 사용 하 여 컨트롤 및 CEBPα/β, 가진, FABP4, leptin, Pref-1, 또는 adiponectin 체형 관련 유전자 서명 (표 1)에 대 한.</li>
	<li>다음 반응 열 cycler에서 실행: 20 95 ° C s 1에 대 한 95 ° C의 40 주기 다음 s 및 20 60 ° C s.</li>
	<li>2-ΔΔCt 메서드를 사용 하 여 배32변경 계산 하.</li>
</ol>

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+microenvironment+alters+protein+expression+and+chemosensitivity+of+epithelial+ovarian+cancer+cells+in+vitro.">A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro.</a> Laboratory Investigation. 93 (5), 528-542 (2013).</li><li>Imamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+2D-+and+3D-culture+models+as+drug-testing+platforms+in+breast+cancer.">Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer.</a> Oncology Reports. 33 (4), 1837-1843 (2015).</li><li>Birgersdotter, A., Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+perturbation+in+vitro--a+growing+case+for+three-dimensional+(3D)+culture+systems.">Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems.</a> Seminars in Cancer Biology. 15 (5), 405-412 (2005).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Dirat, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+adipocytes+exhibit+an+activated+phenotype+and+contribute+to+breast+cancer+invasion.">Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.</a> Cancer Research. 71 (7), 2455-2465 (2011).</li><li>Scott, M. A., Nguyen, V. T., Levi, B., James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+of+adipogenic+differentiation+of+mesenchymal+stem+cells.">Current methods of adipogenic differentiation of mesenchymal stem cells.</a> Stem Cells and Development. 20 (10), 1793-1804 (2011).</li><li>Zipori, D., Toledo, J., von der Mark, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+among+stromal+cell+lines+from+mouse+bone+marrow+disclosed+in+their+extracellular+matrix+composition+and+interactions+with+normal+and+leukemic+cells.">Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells.</a> Blood. 66 (2), 447-455 (1985).</li><li>Maridas, D. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+adipocyte+specification+and+necrosis+augment+tumor-associated+inflammation.">Loss of adipocyte specification and necrosis augment tumor-associated inflammation.</a> Adipocyte. 2 (3), 176-183 (2013).</li><li>Bochet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+adipocytes+promotes+breast+tumor+radioresistance.">Cancer-associated adipocytes promotes breast tumor radioresistance.</a> Biochemical and Biophysical Research Communications. 411 (1), 102-106 (2011).</li><li>Hirano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+adipogenesis+induction+by+conditioned+media+obtained+from+cancer+cells.">Enhancement of adipogenesis induction by conditioned media obtained from cancer cells.</a> Cancer Letters. 268 (2), 286-294 (2008).</li><li>Gordeev, A. 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저자는 선언할 수 없다.

Cocultures 삽입과 셀 상호 작용을 공부 하 널리 사용 되었습니다. 2D coculture 시스템 어떻게 두는 우리가 여기 골 adipocytes에 두 개의 다른 암 세포 기반 효과 보여주는 누화에서 생체 외에서부품을 관찰 하는 효과적인 방법입니다. 많은 연구소는 adipocytes 및 암 세포6,12,,2739사이 누화를 조사 하기 위?...

뼈 전이 고급 암 환자 중 광범위 하지만 치료 치료는 아직 사용할 수 없습니다. 지방으로 에너지를 저장 전문, 넘어 adipocytes 골 수 및 다른 장기1,2,,34,,56에서 종양 성장 및 전이 지원할 수 있습니다. 또한, adipocytes 암 세포 생물학7,,89,10 및 대사4,11,12 조절에 필수적인 역할을 재생 ,13,14,,1516, 잘에서 뼈 전이1,,412로. 골 틈새, adipocytes도 암 세포4,,617의 생물 학적 행동을 달라질 수 있습니다. 골 adipocytes osteotropism와 암 세포 사이 상호 작용에 대 한 뼈 전이의 이해 중요 하다. 그러나, 약간은 알려진 다.
현재 연구를 바탕으로, 다양 한 방법은 adipocytes, 2 또는 3 차원 (2/3 차원)와 비보 전 문화17,,1819,20,21을 포함 하 여 적용 됩니다. 최근, Herroon 그 외 여러분 암 세포22골 adipocytes의 상호 작용을 공부 하는 새로운 3D 문화 접근 방식을 설계 되었습니다. 3D coculture는 생리를 흉내 낸 위한 최적의 adipocytes와 암 사이 상호 작용 세포 vivo에서, 그것은 가난한 재현성22,23에서 고통. 2D coculture 시스템에 비해 3D coculture 시스템 셀 형태학21,22,,2425,26같은 다른 세포질 고기를 제공할 수 있습니다. 또한, 고립 된 수가 뼈 조직 파편의 비보 전 문화 adipocytes의 강력한 파생물 교양된 골 셀17에서 발생할 수 있습니다.
그러나 이러한 이전 모델과 달리, 2D 셀 문화 모델 남아 있다 고전, 안정적이 고 쉽게 기술 생체 외에서1, 세포를 신속 하 게 스캔 후보 분자와 고기 adipocytes 또는 암에 변경에 대 한 4,6,12,,1527. 더 잘 이해 하려면 골 adipocytes와 흑색 종 세포 사이 누화, 우리 흑색 종 세포와 골 adipocytes의 2D coculture 시스템에 대 한 상세한 프로토콜을 제공 합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="685">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:18px;width:215px;">DMEM</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">11965092</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Fetal Bovine Serum</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">16000&#8211;044</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Phosphate Buffered Saline</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">14190-144</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Insulin</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">91077C</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-isobutyl-1-methyl-xanthine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I5879</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Dexamethasone</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">D4902</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Oil Red o</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">O0625</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">24-well plate</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS3527</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Transwell insert</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:143px;">MCHT24H48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:143px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I9516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">0.25% trypsin</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">hemocytometer</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:143px;">1450016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Culture incubator</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">50ml falcon</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS430828</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Clean Bench</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Microscopy</td>
			<td style="width:140px;">Olympus</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">200 &#956;L pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">1000 mL pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP628</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation

골 adipocytes와 종양 세포 사이 누화 뼈 전이 하는 중요 한 역할을 재생할 수 있습니다. 다양 한 방법 공부 하는 중요 한 누화; 사용할 수 있습니다. 그러나, coculture에 대 한 2 차원 transwell 시스템은 고전적인, 신뢰할 수 있는, 그리고이 누화 연구에 대 한 쉬운 방법 남아 있다. 여기, 우리 골 adipocytes 및 흑색 종 세포의 coculture을 보여 주는 자세한 프로토콜을 제시. 그럼에도 불구 하 고, 이러한 coculture 시스템 수 골 adipocytes에 의해 유도 된 암 세포의 세포 신호 transductions의 연구에 기여할 뿐만 아니라 또한 기계적으로 뼈에 대 한 새로운 치료 목표를 드러낼 수 있는 뼈 전이의 연구 전이입니다.

골 수에서 adipocytes 녹는 요인을 통해 종양 진행을 지원 하기 위한 초기 단계에서 종양 microenvironment1,13,33,,3435 에 나타날 수 있습니다 또는 osteoclastogenesis6,,1236, 특히 비만<sup class="x...

Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

여기, 우리 골 adipocytes에 흑색 종 세포 유래 요인의 이중 효과 보여준다 종양 세포와 골 adipocytes, 사이 상호 작용을 공부 하기 위한 안정적이 고 간단한 2 차원 (2D) coculture 시스템 제시 차별화 및 뼈 전이의 기계 론 적인 연구에 대 한 전통적인 방법 포즈.

골 Adipocytes 차별화에 흑색 종 세포 유래 요인의 이중 효과

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

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JoVE Journal

Cancer Research

6.1K 조회수.  Shanghai Jiao Tong University. 여기, 우리 골 adipocytes에 흑색 종 세포 유래 요인의 이중 효과 보여준다 종양 세포와 골 adipocytes, 사이 상호 작용을 공부 하기 위한 안정적이 고 간단한 2 차원 (2D) coculture 시스템 제시 차별화 및 뼈 전이의 기계 론 적인 연구에 대 한 전통적인 방법 포즈.

비디오: Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

암 세포 선 종양 Microenvironment에 Monocytes/대 식 세포의 모집 연구에서 유전자 발현의 조건부 최저

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

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Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

샘플을 측정 하는 측정 가능한 잔여 질환과 급성 골수성 백혈병에 Leukemic 줄기 세포에 대 한 프로세스 골 포괄적인 프로토콜

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D Organotypic 흑색 종 회전 타원 체 피부 모델

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

다 발성 골 수 Xenografts 노 그 쥐에서의 복합 발광 및 양자 방출 단층 촬영/컴퓨터 단층 촬영 영상

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

흑색종 환자 유래 이종이식 모델

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

면역결핍 마우스에서 간 직교 인간 편도 흑색종 Xenograft 플랫폼의 생성

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

고분해능 공초점 현미경검사법에 의한 3D 종양 스페로이드 침습에 대한 Migrastatic 억제제의 특성화

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

투과성 멤브레인 지지대를 이용한 공동 배양을 이용한 대식세포 활성화에 대한 종양 분비 파라크린 리간드의 효과 연구

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

원발성 뼈 종양 및 뼈에 이식된 고형 종양을 통한 뼈 전이 모델링

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

전사 분석을 사용하여 무질서한 온코겐 전사 요인의 구조 기능 관계 매핑

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...