Name: dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation
Uploaded: 2018-08-23T12:30:00
Description: Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

Wij danken Dov Zipori (het Weizmann Institute of Science, Rehovot, Israël) vriendelijk voor het verstrekken van ons de lymfkliertest beenmerg stromale cel 14F1.1 lijn. Deze studie werd ondersteund door subsidies van de Chinese National Natural Science Foundation (Nos. 81771729) en het Yongchuan ziekenhuis van Chongqing medische universiteit (Nos. YJQN201330; YJZQN201527).

Opmerking: Alle cellen die worden gebruikt in dit protocol moeten worden gekweekt voor minstens drie generaties na het ontdooien van bevroren voorraad cellen.
1. de oogst van melanoom cel-afgeleide factoren
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Voorbereidingen
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<li>B16F10 cellen en een muis melanoom cellijn verkrijgen. Opmerking: Voor dit protocol, een muis melanoom cellijn werd verkregen van de Bank van de cel van de stam van de Chinese Academie van Wetenschappen.</li>
		<li>Maak een volledige ...

<ol>
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Cocultures met inzetstukken hebben wijd gebruikt om te studeren van cel naar cel interacties. Het 2D coculture-systeem is een effectieve manier om te observeren hoe de twee delen Overspraak in vitro, waaraan we hier twee verschillende kanker cel-gedreven effecten op het beenmerg adipocytes toonden. Veel laboratoria hebben gebruikt deze methode om te onderzoeken de Overspraak tussen adipocytes en kanker cellen6,12,27<sup...

Botmetastasen zijn wijdverspreid onder Geavanceerde kankerpatiënten, maar een curatieve behandeling is nog steeds niet beschikbaar. Verder gespecialiseerd in energie op te slaan als vet, kan adipocytes ondersteunen tumorgroei en uitzaaiing in het beenmerg en andere organen1,2,3,4,5,6 Bovendien spelen adipocytes een essentiële rol bij het reguleren van kanker cel biologie7,8,9,10 en metabolisme4,11,12 ,13,14,15,16, zo goed als in bot metastase1,4,12. In de niche van het beenmerg, adipocytes kan ook invloed hebben op de biologische werking van kanker cellen,4,,6,17. Het samenspel tussen het beenmerg adipocytes en kankercellen met osteotropism is belangrijk voor een goed begrip van bot metastase. Echter, er is weinig bekend.
Op basis van de huidige studies, worden verschillende methoden toegepast op adipocytes, met inbegrip van twee - of drie - dimensional (2/3D) en ex vivo culturen17,18,19,20,21. Herroon et al. ontworpen onlangs, een nieuwe aanpak van het 3D-cultuur te bestuderen van interacties van beenmerg adipocytes met kanker cellen22. Hoewel de 3D coculture is optimaal voor het nabootsen van fysiologische interacties tussen adipocytes en kanker cellen in vivo, het lijdt aan slechte reproduceerbaarheid22,23. In vergelijking met een 2D coculture-systeem kunnen een 3D coculture-systeem verschillende cellulaire fenotypen, zoals cel morfologie21,22,24,25,26. Bovendien, de ex vivo cultuur van geïsoleerde spongieuze weefsel fragmenten kan leiden tot een robuuste uitvloeisel van adipocytes van gekweekte beenmerg cellen17.
In tegenstelling tot deze eerdere modellen blijft de 2D cell cultuur model echter een klassieke, betrouwbare en gemakkelijke techniek voor snel scannen van kandidaat-moleculen en de fenotypes veranderd in adipocytes of kanker in vitro1cellen, 4,6,12,15,27. Om beter te begrijpen de Overspraak tussen beenmerg adipocytes en melanoom cellen, bieden wij een gedetailleerd protocol voor een 2D coculture-systeem van het beenmerg adipocytes met melanoom cellen.

<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

De Overspraak beenmerg adipocytes à tumorcellen kan spelen een cruciale rol in het proces van bot metastase. Een verscheidenheid van methoden zijn beschikbaar voor de studie van de significante Overspraak; een twee-dimensionale transwell systeem voor coculture blijft echter een klassiek, betrouwbaar, en gemakkelijke manier voor deze studie overspraak. Hier presenteren we een gedetailleerd protocol waarin de coculture van het beenmerg adipocytes en melanoom cellen. Echter zou zulk een coculture systeem niet alleen bijdragen tot de studie van cel signaal transductions van kankercellen geïnduceerd door het beenmerg adipocytes, maar ook naar de toekomst mechanistische studie van bot metastase, die nieuwe therapeutische streefcijfers voor bot kan onthullen metastase.

In het beenmerg, adipocytes kunnen worden weergegeven in de tumor communicatie1,13,33,34,35 in een vroeg stadium voor de ondersteuning van de tumor progressie door oplosbare factoren of het activeren van osteoclastogenesis6,12,36, met ...

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

Hier presenteren we een betrouwbare en eenvoudige tweedimensionale (2D) coculture-systeem voor de studie van de interactie tussen tumorcellen en beenmerg adipocytes, die een dubbele effect van melanoom cel-afgeleide factoren op het beenmerg-adipocytes onthult differentiatie en vormt ook een klassieke methode voor de mechanistische studie van bot metastase.

Dual effecten van melanoom cel-afgeleide factoren op het beenmerg Adipocytes differentiatie

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 ...

Research

JoVE Journal

Cancer Research

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

Voorwaardelijke Knockdown van genexpressie in kanker cellijnen te bestuderen van de aanwerving van monocyten/macrofagen naar de Tumor communicatie

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

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

Uitgebreide Protocol bij monster en proces beenmerg voor het meten van meetbare residuele ziekte en leukemische cellen van de stam in Acute myeloïde leukemie

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

Een 3D Organotypic melanoom sferoïde huidmodel

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

Multimodale bioluminescente en positronisch-emissie tomografie/computationele Tomography Imaging van Multiple Myeloma beenmerg Xenografts in NOG muizen

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

Een melanoom patiënt-afgeleide xenograft model

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

Generatie van een lever Orthotopic humaan Ukalfs melanoom xenograft platform in Immunodeficiënte muizen

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

Karakterisering van de effecten van migraine remmers op de invasie van 3D-tumor Spheroid door confocale microscopie met hoge resolutie

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

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

Modellering van primaire bottumoren en botmetastase met implantatie van solide tumortransplantaten in bot

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

Het in kaart brengen van de structuur-functierelaties van ongeordende oncogene transcriptiefactoren met behulp van transcriptomische analyse

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

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

Modelleren van de effecten van hemodynamische stress op circulerende tumorcellen met behulp van een spuit en naald

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...