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

Vi takker Dov Zipori (The Weizmann Institute of Science, Rehovot, Israel) venligst for at give os murine knoglemarv stromale celle linje 14F1.1. Denne undersøgelse blev støttet af tilskud fra den kinesiske National Natural Science Foundation (nr. 81771729) og det Yongchuan Hospital i Chongqing medicinske universitet (nr. YJQN201330; YJZQN201527).

Bemærk: Alle celler, der bruges i denne protokol skal være dyrket i mindst tre generationer efter optøning af frosne stock celler.
1. høst melanom celle-afledte faktorer
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Præparater
	<ol>
<li>Få B16F10 celler og en mus melanom cell line. Bemærk: Til denne protokol, en mus melanom cell line blev indhentet fra stamcelle Bank af kinesiske Academy of Sciences.</li>
		<li>Gøre en komplet medium for B16F10 cellekultur (100 mL). Bruge Dulbeccos modificerede Eagle...

<ol>
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Cocultures med skær har været udbredt at studere celle-til-celle interaktioner. 2D coculture system er en effektiv måde at iagttage, hvordan de to dele krydstale in vitro-, som vi her viste to forskellige kræft celle-drevet effekter på knoglemarv adipocytter. Mange laboratorier har udnyttet denne metode til at undersøge krydstale mellem adipocytter og kræft celler6,12,27,39....

Knoglemetastaser er udbredt blandt fremskreden kræftpatienter, men en kurativ behandling er stadig ikke tilgængelig. Ud over med speciale i lagre energi som fedt, kan adipocytter støtte tumorvækst og metastase i knoglemarven og andre organer1,2,3,4,5,6. Derudover spiller adipocytter en vigtig rolle i reguleringen af kræft celle biologi7,8,9,10 og metabolisme4,11,12 ,13,14,15,16, som godt som i knogle metastaser1,4,12. I knoglemarven niche, kan adipocytter også påvirke den biologiske opførsel af kræft celler4,6,17. Samspillet mellem knoglemarv adipocytter og kræftceller med osteotropism er væsentlig for forståelsen af knogle metastaser. Dog er lidt kendt.
Baseret på de aktuelle undersøgelser, der forskellige metoder anvendes på adipocytter, herunder to - eller tre - dimensionelle (2/3D) og ex vivo kulturer17,18,19,20,21. For nylig, Herroon et al. designet en ny 3D-kultur tilgang til at studere samspillet mellem knoglemarv adipocytter med kræft celler22. Selv om den 3D coculture er optimal for efterligne fysiologiske interaktioner mellem adipocytter og kræft celler, i vivo, det lider af dårlig reproducerbarhed22,23. Sammenlignet med en 2D coculture system, kan en 3D coculture system give forskellige cellulære fænotyper, såsom celle morfologi21,22,24,25,26. Desuden kan ex vivo kultur af isolerede spongiosa væv fragmenter føre til en robust udvækst af adipocytter fra kulturperler knoglemarv celler17.
I modsætning til disse tidligere modeller, men stadig 2D celle kultur model en klassisk, pålidelig og let teknik til hurtigt scanning kandidat molekyler og fænotyper ændres i enten adipocytter eller kræft celler in vitro-1, 4,6,12,15,27. For bedre at forstå krydstale mellem knoglemarv adipocytter og melanom celler, giver vi en detaljeret protokol for en 2D coculture system af knoglemarven adipocytter med melanom celler.

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	<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

Krydstale mellem knoglemarv adipocytter og tumorceller kan spille en kritisk rolle i knogle metastaser. En række forskellige metoder er tilgængelige til at studere den betydelige krydstale; en to-dimensional transwell system for coculture er imidlertid fortsat en klassisk, pålidelig og let måde for denne krydstale undersøgelse. Vi præsenterer her, en detaljeret protokol, der viser coculture af knoglemarven adipocytter og melanom celler. Men sådan et coculture system kunne ikke kun bidrage til studie af celle signal transductions af kræftceller induceret af knoglemarven adipocytter, men også til fremtidens mekanistisk undersøgelse af knogle metastaser, der kan afsløre nye terapeutiske mål for knogle metastaser.

I knoglemarven, adipocytter kan vises i tumor mikromiljø1,13,33,34,35 på et tidligt tidspunkt for at støtte tumor progression gennem opløselige faktorer eller aktivering osteoclastogenesis6,12,36, især i forbindelse med fedme<sup ...

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

Her præsenterer vi en pålidelig og enkel todimensionale (2D) coculture system til at studere samspillet mellem tumorceller og knoglemarv adipocytter, hvilket afslører en dobbelt effekt af melanom celle-afledte faktorer på knoglemarv adipocytter differentiering og udgør også en klassisk metode for mekanistiske studier af knogle metastaser.

Dobbelt virkningerne af melanom celle-afledte faktorer på knoglemarv adipocytter differentiering

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

Betinget Knockdown af genekspression i kræft cellelinjer til at studere ansættelse af monocytter/makrofager at Tumor mikromiljø

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

Omfattende protokol til prøven og proces knoglemarven til at måle målbare Residual sygdom og Leukemic stamceller i akut Myeloid leukæmi

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

En 3D Organotypic melanom klumpformet hudmodel

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

Multimodal en bioluminescerende og Positron-emissions-tomografi/edb tomografi billeddannelse af myelomatose knoglemarv Xenografts i NOG mus

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

En melanom patient afledt 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

Generation af en lever Orthotopic humant Ukalvekød melanom xenograft platform i immundefekt mus

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 af virkningerne af Migrastatiske hæmmere på 3D tumor Spheroid invasion af høj opløsning Konfokal mikroskopi

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 af primære knogletumorer og knoglemetastase med implantation af fast tumortransplantat i knogle

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

Kortlægning af strukturfunktionsforhold for uordnede onkogene transskriptionsfaktorer ved hjælp af transskriptomisk 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

Modellering virkningerne af hæmodynamisk stress på cirkulerende tumorceller ved hjælp af en sprøjte og nål

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