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-Instituttet for vitenskap, Rehovot, Israel) ber for å gi oss murine benmarg stromal cellen linje 14F1.1. Denne studien ble støttet av tilskudd fra kinesiske National Natural Science Foundation (nr. 81771729) og Yongchuan Hospital i Chongqing medisinske universitet (Nos. YJQN201330; YJZQN201527).

Merk: Alle cellene som brukes i denne protokollen må være dyrket i minst tre generasjoner etter tining av frosne lager celler.
1. harvest melanom Cell-derived faktorer
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Forberedelser
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<li>Få B16F10 celler og en mus melanom cellen linje. Merk: For denne protokollen, en mus melanom cellen linje er Hentet fra den stilk cellen Bank av det kinesiske vitenskapsakademi.</li>
		<li>Foreta et komplett medium for B16F10 cellekultur (100 mL). Bruk Dulbeccos endret Ea...

<ol>
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Cocultures med innlegg har vært mye brukt til å studere celle til celle interaksjoner. 2D coculture systemet er en effektiv måte å observere hvordan de to delene crosstalk i vitro, som vi her viste to forskjellige kreft celle-drevet effekter på benmarg adipocytter. Mange labs har brukt denne metoden for å undersøke crosstalk mellom adipocytter og kreft celler6,12,27,39.
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Benmetastaser er utbredt blant avansert kreftpasienter, men en helbredende behandling er fremdeles utilgjengelig. Utover spesialiserer seg på lagring av energi som fett, kan adipocytter støtte tumor vekst og metastasering i benmargen og andre organer1,2,3,4,5,6. Videre spiller adipocytter en viktig rolle i å regulere kreft cellen biologi7,8,9,10 og metabolisme4,11,12 ,13,14,15,16, så vel som i bein metastasering1,4,12. I benmargen nisje, kan adipocytter også påvirke biologisk atferd av kreft celler4,6,17. Samspillet mellom benmarg adipocytter og kreftceller med osteotropism er viktig for en forståelse av bein metastasering. Men er lite kjent.
Basert på dagens studier, brukes ulike metoder på adipocytter, inkludert to - eller tre - dimensjonale (2/3D) og ex vivo kulturer17,18,19,20,21. Nylig utviklet Herroon et al. en ny 3D-kultur tilnærming til studere interaksjoner av benmarg adipocytter med kreft celler22. Selv om det 3D coculture er optimal for mimicking fysiologiske interaksjoner mellom adipocytter og kreft celler i vivo, den lider av dårlig reproduserbarhet22,23. I forhold til en 2D coculture system, kan en 3D coculture-system gi mobilnettet Norge, for eksempel cellen morfologi21,22,24,25,26. Videre kan ex vivo kulturen av isolerte cancellous bein vev føre til en robust utvekst av adipocytter kulturperler bein margtransplantasjon celler17.
I motsetning til disse tidligere modeller imidlertid 2D celle kultur modellen en klassisk, pålitelig og enkel teknikk for raskt skanne kandidat molekyler og av fenotyper i enten adipocytter eller kreft celler i vitro1, 4,6,12,15,27. For bedre å forstå crosstalk mellom benmarg adipocytter og melanom celler, gir vi en detaljert protokoll for et 2D coculture system av benmarg adipocytter med melanom celler.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="685">
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		<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

Crosstalk mellom benmarg adipocytter og kreftceller kan spille en avgjørende rolle i prosessen med bein metastasering. En rekke metoder er tilgjengelige for å studere den betydelige crosstalk; imidlertid fortsatt en todimensjonal transwell system for coculture en klassisk, pålitelig, og enkel måte for denne crosstalk studien. Her presenterer vi en detaljert protokoll som viser coculture av benmarg adipocytter og melanom celler. Likevel kunne slikt coculture system ikke bare bidra til studiet av cellen signal transductions av kreftceller av benmarg adipocytter, men også i fremtiden mekanistisk studie av bein metastasering som kan avsløre nye terapeutiske mål for bein metastasering.

I benmargen, adipocytter kan vises i de svulst microenvironment1,13,33,34,35 på et tidlig stadium for å støtte svulst progresjon gjennom løselig faktorer eller aktivere osteoclastogenesis6,12,36, spesielt i sammenheng 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 presenterer vi en pålitelig og enkel todimensjonal (2D) coculture system for å studere samspillet mellom kreftceller og benmarg adipocytter, som avslører en dobbel effekt av melanom celle-avledet faktorer på benmarg adipocytter differensiering og utgjør også en klassisk metode for mekanistisk studier av bein metastasering.

To effekter av melanom Cell-derived faktorer på benmarg adipocytter differensiering

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 av genuttrykk i kreftcelle linjer å studere rekruttering av monocytter/makrofager til Tumor Microenvironment

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 protokoll for å prøve og prosessen benmarg for å måle målbare gjenværende sykdom og Leukemic stamceller i akutt myelogen leukemi

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

3D Organotypic melanom Spheroid huden modell

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

Flere Bioluminescent og Positronic-utslipp tomografi/beregningsorientert tomografi avbilding av myelom benmarg 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 pasient-avledet Xenograft modell

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

Generering av en Liver Orthotopic Human uveal melanom Xenograft Platform i Immunodeficient 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 av effekter av Migrastatic hemmere på 3D tumor spheroid Invasion av høy oppløsning Konfokalmikroskopi 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 av primære beintumorer og benmetastase med implantasjon av solid tumortransplantat i bein

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

Kartlegging av strukturfunksjonsrelasjoner av uordnede onkogene transkripsjonsfaktorer ved hjelp av transkripsjonsanalyse

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 av effekten av hemodynamisk stress på sirkulerende tumorceller ved hjelp av en sprøyte og nål

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