Name: analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system
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Description: Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Dette arbejde blev støttet af CONACyT FONSEC SSA/IMSS/ISSSTE projekt nr. 233061 til Ezequiel M. Fuentes-Pananá og af Fondo de Apoyo a la Investigación, Hospital Infantil de México Federico Gómez (projektnummer HIM-2014-053). Espinoza-Sánchez NA er en ph.d.-studerende fra Programa de Doctorado da Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) og modtaget stipendium 231663 fra CONACYT. E-S NA, også anerkende den finansielle støtte fra det mexicanske Institut for socialsikring (IMSS).

Prøver fra BrC patienter blev indhentet fra vævsbank Unidad de Investigación eget Virología y Cáncer, Hospital Infantil de México Federico Gómez. Denne undersøgelse blev godkendt af den videnskabelige, etiske, og biosikkerhedsforanstaltninger review bestyrelserne for Hospital Infantil de México Federico Gómez: Comité de Investigación, Comité de Ética da Investigación og Comité de Bioseguridad. Alle patienter blev prospektivt indskrevet og blev informeret om karakteren af undersøgelsen: der er villige til...

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Epitelceller vokser i en 3D fysisk kropsbygning og deres interaktion med ECM proteiner er afgørende for væv homøostase. Mange kræft undersøgelser har været baseret på celler dyrkes i encellelag (2D) og selv om de har været afgørende for at forstå mange aspekter af tumordannelse og progression, encellelag sammenfatte ikke de karakteristika, der ECM pålægger celler, for forekomst: begrænse spredning, vedhæftning-afhængige celle overlevelse, apikale basolateral polaritet, ECM remodeling, Celledifferentiering,...

Tumor biologi er langt mere indviklet end hidtil antaget. Montering beviser viser, at tumorceller er mere end en simpel bulk af ukontrolleret prolifererende celler; Tværtimod synes forskellige neoplastiske celler til at udføre forskellige funktioner viser høj organisation og hierarki inden for tumor1. Tumorceller er også i intime kommunikation med ikke-transformeret celler: makrofager, fibroblaster, lymfocytter, adipocytter, endotelceller, blandt andre celler er alle nedsænket i stillads proteiner og polysaccharider, der udgør ECM. Mange direkte og indirekte vekselvirkninger er etableret mellem transformeret og ikke-transformeret celler og med ECM, som udøver en kraftig indflydelse på sygdommen resultatet2,3. I det konkrete tilfælde med BrC er det særlig vigtigt at dissekere meddelelse af BrC celler med TAMs, overvejer at TAMs har vist sig for at spille en afgørende rolle i udviklingen af tumor, øge risikoen for metastaser og for sygdom tilbagefald4 ,5.
For at analysere samhandel tumorsygdomme interaktioner og deres mulige udfald, nye 3D in vitro- metoder er blevet udviklet baseret på brugen af ECM ekstrakter, der giver en langt mere kompleks mikromiljø, tættere på virkeligheden af tumor biology, i sammenligning med de konventionelle éncellelag cellekulturer som celler vokse fastgjort til plastik. Petersen og Bissell6 leveret den første model af nonmalignant, og ondartet brystkirtler epitelceller kulturperler på en laminin-rige basalmembranen og var de første til at beskrive de 3D organotypic strukturer, der diskriminerer nonmalignant menneskelige bryst epitelceller fra deres maligne modstykker. Et årti senere, modellen udviklet af Debnath, Muthuswamy, og Brugge7,8,9 leveret et værdifuldt redskab for at belyse den biologiske veje i fare under maligne transformation af glandulær acini, sådan som store acini dannelse på grund af ukontrolleret spredning, virksomhedsflytninger af stram vejkryds proteiner som bevis for nedsat celle polarisering, og tab af acini lumen som følge af celle modstand mod anoikis, en type af programmeret celledød, der opstår i forankring-afhængige celler når de løsne sig fra de omkringliggende ECM. Modeller af Sameni, Jedeszko og Sloane har fokuseret på imaging proteolytiske aktivitet af celler, som er nært beslægtet med invasiv, en anden afgørende træk af tumor malignitet10,11,12. Disse modeller er afhængige af protein matricer blandet med forskellige fluorescens-bratkølet protein substrater (DQ-gelatine, DQ-kollagen I, og DQ-kollagen IV), i hvilke fluorescerende signaler er vejledende for den proteolytisk nedbrydning af kollagen. 3D-modeller er også bruges til at undersøge stamceller egenskaber af både ikke-transformeret og tumorceller, i hvilken celle aggregater, også kaldt spheroids, kan blive kulturperler i suspension eller i ECM-lignende proteiner spørgekriterierne for mekanismer af Celledifferentiering, asymmetrisk celledeling, celle til celle overholdelse og celle motilitet13,14. Invasion assays giver mulighed for prøvning af den iboende aggressivitet af tumor og identifikation af de molekyler, der tjener som chemoattractants under invasive proces15. Samlede, 3D modeller repræsenterer en overkommelig diversificering af in vitro- cellekultur, der mere nøje afspejler normale og muterede væv morfogenese.
Vi har designet en 3D celle Co kultur system baseret på ovennævnte modeller7,10,11ved hjælp af både menneskelige kommercielle BrC cellelinjer kendte aggressive potentiale (luminale og triple-negativ typer) og primære celler eksplanterede fra BrC patienter. Vi først udviklet en model hvor enten ikke-aggressive (MCF-7) eller aggressive (MDA-MB-231) BrC celler var Co kulturperler med U937 monocytter i en ekstracellulære matrix ekstrakt (ECME)-baseret 3D system, der tillod direkte celle-celle interaktioner. Disse fælles kulturer blev brugt til at bestemme, hvordan kommunikationen mellem disse to celle lineages påvirket transskription af et sæt af gener relateret til kræft aggressiv adfærd. En væsentlig forøgelse af cyclooxygenase-2 (COX-2) udskrift konstateredes, faldt sammen med en øget produktion af en af sine produkter, prostaglandin E2 (PGE2), en konstatering af, at fremhævet rollen betændelse i kræft progression. Øget transkription af MMP blev også observeret der korreleret med større kollagen proteolyse når aggressive MDA-MB-231 celler var Co kulturperler med U937 monocytter i DQ-kollagen IV-holdige kulturer. Bemærk, vores co kulturer ikke understøtter den antagelse, at celle-celle interaktion mekanismer er nødvendig for kollagen nedbrydning. Det foreslog snarere, at kommunikation mellem to celle lineages blev formidlet af udskilles molekyler. Derudover indeholdt analysere høstet fra disse fælles kultur assays opløselige faktorer, der uorganiseret glandulær acini dannet af ikke-transformeret MCF-10A celler13. Det blev fundet, aggressive og primære BrC celler udskilles forhøjede niveauer af monocyt kemotaktisk molekyler MCP-1, GM-CSF og RANTES. Dermed, vi skitseret en 3D kultur hvor celler var adskilt i celle kultur skær at forhindre celle-celle interaktioner. Disse kulturer blev brugt til at behandle den indirekte kommunikation mellem BrC celler og monocytter. For disse assays, ikke-aggressiv og pågående kommercielle BrC cellelinjer og primære BrC celler og tre forskellige typer af humane monocytter: kommercielle U937 og THP-1 celler og primære monocytter (PMs) isoleret fra perifert blod af raske donorer, (alle monocytter blev brugt i en ikke-aktiveret tilstand) blev brugt. Øgede koncentrationer af inflammatoriske cytokiner IL-1β og IL-8 blev observeret at være beriget med fælles kultur. Ligeledes blev det konstateret at MMP-1, MMP-2 og MMP-10 var også øget i BrC celler-monocyt Co kulturer og således styrke tidligere resultater14. I dette manuskript præsenteres en punkt for punkt arbejdsproces af primære BrC celler isolation og test i 3D kulturer sammen med repræsentative resultater. Dette arbejde udgør et godt eksempel på det store potentiale, som in vitro- 3D celle systemer giver for at afhøre specifikke aspekter af tumor biology.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">U937</td>
			<td style="width:260px;">American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">CRL-1593.2</td>
			<td style="width:463px;">Monocytic cell line/histiocytic lymphoma&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">THP-1</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">TIB-202</td>
			<td>Monocytic cell line/acute monocytic leukemia&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-10A</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td>CRL-10317</td>
			<td>Non-transformed breast cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCF-7</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-22</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">T47D</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-133</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HS578T</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-126</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MDA-MB-231</td>
			<td>American Type Culture Collection ATCC&#160;</td>
			<td style="width:143px;">HTB-26</td>
			<td>Breast Cancer Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RPMI 1640 medium</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM High Glucose&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11964-092</td>
			<td>4.5 g/L glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEM/F12</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">11039-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Antibiotic/Antimycotic</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">15240-062</td>
			<td>100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 0.25 &#181;g/mL Fungizone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Horse serum&#160;</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>16050114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin-EDTA 1X</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td style="width:143px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X (Phosphate Buffered Saline</td>
			<td style="width:260px;">GIBCO BRL Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>AF-100-15 (1.00mg)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I1882-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>SIGMA-ALDRICH</td>
			<td>H088-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholera toxin Vibrio cholerae</td>
			<td>SIGMA-ALDRICH</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Matrigel&#160;</td>
			<td>Corning Inc</td>
			<td>356237</td>
			<td style="width:463px;">Engelbreth-Holm-Swarm (EHS) mouse sarcoma, extracellular matrix extract, Store at -20&#176;C until use at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">GM-CSF</td>
			<td>PeproTech</td>
			<td>300-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">MCP-1</td>
			<td>PeproTech</td>
			<td>300-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">RANTES</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-8</td>
			<td>PeproTech</td>
			<td>200-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IL-1&#946;</td>
			<td>PeproTech</td>
			<td>200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Crystal-violet</td>
			<td>Hycel M&#233;xico</td>
			<td>541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Corning Inc</td>
			<td style="width:143px;">3422</td>
			<td>6.5 mm diameter, 8 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transwell permeable supports (inserts)</td>
			<td>Thermofisher Scientific</td>
			<td style="width:143px;">140620</td>
			<td>6.5 mm diameter, 0.4 &#181;m pore size/24-well plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Monocyte Isolation Kit II Human&#160;</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-091-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LS Columns</td>
			<td style="width:260px;">Miltenyi Biotec</td>
			<td style="width:143px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Human Cytokine/Chemokine Magnetic Bead Panel Kit 96 Well Plate Assay</td>
			<td style="width:260px;">EMD Millipore</td>
			<td style="width:143px;">HCYTOMAG-60K</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Magpix with xPonent software (laser based fluorescent analytical test instrumentation)</td>
			<td style="width:260px;">Luminex Corporation</td>
			<td style="width:143px;">40-072</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Lab-Tek chamber slide with cover (8 well)</td>
			<td style="width:260px;">Nalge Nunc International</td>
			<td style="width:143px;">177402</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Glass bottom microwell 35mm petri&#160; dishes</td>
			<td style="width:260px;">MatTek Corp.</td>
			<td style="width:143px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the communication between monocytes and primary breast cancer cells in an extracellular matrix extract (ecme)-based three-dimensional system

Indlejret i den ekstracellulære matrix (ECM), kommunikere normale og neoplastiske epitelceller nøje med hæmatopoietisk og ikke-hæmatopoietisk celler, således betydeligt påvirke normale væv homøostase og sygdom resultat. I brystkræft spiller tumor-associerede makrofager (TAMs) en afgørende rolle i sygdomsprogression, metastaser og gentagelse; derfor forstå mekanismer af monocyt chemoattraction tumor mikromiljø og deres interaktioner med tumorceller er vigtige til bekæmpelse af sygdommen. Her, giver vi en detaljeret beskrivelse af en tredimensional (3D) fælles kultur system af menneskelig brystkræft (BrC) celler og humane monocytter. BrC celler produceret høj basal niveauer af regulerede på aktivering, normal T-celle udtrykt og udskilles (RANTES), monocyt chemoattractant protein-1 (MCP-1) og granulocyt-makrofag koloni-stimulerende faktor (G-CSF), mens der i fælles kultur med monocytter, pro-inflammatoriske cytokiner Interleukin (IL) -1 beta (IL-1β) og IL-8 blev beriget med matrix metalloproteinases (MMP) -1, MMP-2 og MMP-10. Denne tumor stroma mikromiljø forfremmet modstand mod anoikis i MCF-10A 3D acini-lignende strukturer, chemoattraction af monocytter og invasion af aggressive BrC celler. Protokollerne præsenteres her giver en overkommelig alternativ til at studere intra-tumor kommunikation og er et eksempel på det store potentiale, som in vitro- 3D celle systemer giver for at afhøre specifikke funktioner for tumor biology relateret til tumor aggression.

Morfologisk analyse af BrC celler i 3D kulturer:
Morfologi af primære BrC celler vokser i 3D kulturer ved lave og høje tætheder blev studeret over 5 dage. Under de første 48 timer, celler overholder ECME og fastholde en lav befolkningstæthed. På dette tidspunkt, kan det klart være værdsat, at celler præsentere et aflangt spindel-lignende form, nogle med to eller flere lange cytoplasmatisk fremskrivninge...

Watch this Scientific Journal Video about Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System at JoVE.c

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Analysere kommunikationen mellem monocytter og primær brystkræftceller i en ekstracellulære Matrix ekstrakt (ECME)-baseret tre-dimensionelle System

Her beskriver vi en tre-dimensionel kultur metode til at analysere morfologi af primær brystkræftceller, samt at studere deres direkte/indirekte interaktioner med monocytter og resultater som kollagen nedbrydning, immun celle rekruttering, celle invasion, og fremme af cancer-relaterede inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Research

JoVE Journal

Cancer Research

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Ved hjælp af Functional Genomics Screening at identificere potentielt nyt lægemiddel mod cancer Cell sfæroide Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Bevæger sig opad: En enkel og fleksibel In Vitro tredimensionale Invasion Assay protokol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

En prækliniske musemodel af osteosarkom at definere den ekstracellulære vesikel-medieret kommunikation mellem Tumor og mesenkymale stamceller

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

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

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

Fabrikation af tungen ekstracellulære Matrix og rekonstituering af tungen planocellulært karcinom In Vitro

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

En tredimensionel Thymisk kultur system til at generere murine induceret pluripotente stamcelle-afledte tumor antigen-specifikke Thymisk emigranter

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Tre-dimensionelle knogle ekstracellulære Matrix Model for osteosarkom

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

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Indfange små molekyle kommunikation mellem væv og celler ved hjælp af Imaging massespektrometri

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Fuldt humant tumor-baserede matrix i tredimensionale Spheroid invasion assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Differentiering af mus bryst epitel HC11 og EpH4 Celler

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Modellering af brystkræft i humant brystvæv ved hjælp af et mikrofysiologisk system

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...