Name: flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells
Uploaded: 2018-01-10T14:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells at JoVE.

이 작품은 NIH R01CA187780에 의해 부분적으로 지원 되었습니다.

참고: Astroglial 및 신경 lentivirus 기자 시스템 사전 패키지, 집중 lentiviral 준비로 구매 되었다. 교류 cytometry 기술의 기본 지식이 필요 합니다. 또한,이 프로토콜을 사용 하는 전체에 대 한 사용자 흐름 cytometer 높은 처리량 용량 (샘플 소스로 96 잘 접시를 허용)에 대 한 액세스를 해야 합니다.
1. lentiviral Transcriptional 기자 시스템
참고: 모든 흐름 cytometric 분석에 사...

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그들을 정의 하는 표식에 주로 집중 하는 GSCs의 이전 연구의 대부분,이 연구에서 우리가 결정 역 접근 하는 것. 우리는 GSCs (예를 들어, 셀 astroglial와 신경 마커)에 의해 생성 된 차별화 된 progenies에 주로 집중 한다. 여기 우리 인간의 GFAP 발기인 종속 식을 GFP의 기반 시스템 심사 높은 처리량 셀 기반 약물의 사용을 보여 줍니다. 모든 실험 환자 파생 neurosphere 세포종 세포 라인을 활용 하 여 수행 했다...

최근 연구는 작은 인구를 포함 하는 종양 세포, 암 줄기 세포 (CSCs) 또는 시작 하는 종양 세포 나의 종양 진행, 전이, 그리고 항 암 치료 및 라디오 치료 1, 저항에 대 한 책임은 2. 암 줄기 세포 및 종양 내의 그들의 분화 progenies intratumoral이 추진 하는 중요 한 요소를 간주 하 고 따라서 암3치료에 주요 장애물을 나타냅니다. 종양 세포 계층, 암 줄기 세포 이론에 의해 제공 된 치료 암 4새로운 전략의 개발 영감 하고있다. 암 줄기 세포를 대상으로 하는 한 가지 방법은 식별 하 고 영향을 받는 장기의 배아 발달 동안에 필요한 것으로 알려져 있습니다 신호 경로 억제 하는 것입니다. 사실, 우리와 다른 사람 신경 줄기 세포 관련 신호 경로 소닉 고슴도치와 노치 세포종5,,67에 대 한 지속적인 요구를 설명 하는 여러 논문을 출판 이전 했습니다. 이 작품은 여러 GBM 임상 시험에 대 한 근거를 응고에 도움이 되었습니다. 암 줄기 세포를 대상으로 두 번째 방법은 그들의 차별화 홍보 하는 것입니다. 이 방법은 retinoic 산 (ATRA, 비타민 A 아날로그)와 급성 promyelocytic 백혈병 치료에 전 임상 및 임상 연구에서 유리한 결과로 인해 지원 많이 받았습니다. 여기 ATRA 성숙 블록을 제거 하 고 암 세포 감 별 법8촉진 발견 됐다. 더 최근에, Piccirillo와 동료 우아하게 나타났습니다 BMP-4 중요 한 안티-GBM 효과 생체 외에서 그리고 vivo에서9이다에 GSC 차별화를 촉진.
현재 연구에 대 한 근거 GSCs를 대상으로 "역된 공학" 접근을 기반으로 합니다. GBM와 암의 특징 중 하나 되 고 불 쌍 한 차별화와 광대 한이 감안할 때, 우리는 경우 우리는 호의 베푸는 표현 형-사이토와 같은 상태로 차별화 홍보 수 물었다. 여기, 우리가 특정된 종양 견본에서 GSCs를 유지 하지만 오히려 원하는 표현 형 (예: GFAP 양성) 달성을 목표로 하는 신호 통로의 사전 지식이 필요가 없습니다.
이 보고서는 GSC 차별화 기자-라인 GSC 농축 문화 변환에서 GSC 클론 선택 영역을 설정 하는 데 사용 하는 절차를 설명 합니다. 세포종 neurosphere 라인 사용 병원 산 라파엘 레-밀라노, 이탈리아에서 기본 세포종의 진단 가진 환자에서 교수 안젤로 Vescovi의 실험실에서 설립 되었다. 이 라인은 여러 간행물 6,10,,1112,13,14에서 광범위 하 게 연구 되었습니다. 그들의 실험실에서 이러한 기술을 구현에 관심이 있는 개인 공부를 계획 하는 세포에서 암 줄기 세포 자체 갱신 용량 기자의 관련성 확인 것이 좋습니다 (이 어떤 기자). 생체 외에서 clonogenic 분석 실험은 분야에서 허용 중 하나에 대 한 자세한 프로토콜이15,16을 달성 하기 위해 제공 됩니다. 마지막으로, cytometry 기반 약 스크린에 차별화 기자-라인의 활용을 설명 하는 상세한 프로토콜 끝에 제공 됩니다. 노트, 마찬가지로 astroglial 차별화 시스템 설명, 우리 성공적으로 설립 하 고 GSC 기자 라인 MAP2:GFP (신경 분화) 기자를 통합 검증. 따라서,이 방법론 설명 종이 다양 한 세포 계보에 세포 분화 연구에 적용할 수 있습니다.
이 보고서에 있는 숫자 중 일부는 최근 간행물에서 찾을 수 있습니다: "Atracurium Besylate 다른 신경 근육 차단 에이전트 astroglial 차별화 홍보 및 세포종 줄기 세포18고갈.

<table>
	<tbody>
		<tr>
			<td>ESGRO Complete Accutase</td>
			<td>EMD Millipore</td>
			<td>SF006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (Hank&#39;s Balanced Salt Solution)</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GFAP Differentiation Reporter (pGreenZeo, Virus)</td>
			<td>SBI (System Biosciences)</td>
			<td>SR10015VA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile disposable reagent reservoirs</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T25 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T75 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130190</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 0.5 - 20uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 5-250uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 25 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 250 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-9025</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava easyCyte 5HT Benchtop Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>0500-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH Clinical Collections 1 and 2 small molecule libraries</td>
			<td>Evotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For the preparation of neural stem cell media (500 mL)</td>
			<td></td>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBio.com</td>
			<td>A-421-250</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>DMEM/F12 10X</td>
			<td>Corning</td>
			<td>90-091-PB</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td>0.0002%</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Sigma Aldrich</td>
			<td>H4036</td>
			<td>5.4 mM</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin- Selenium (ITS -G) (100X)</td>
			<td>Life Technologies</td>
			<td>41400-045</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td>14.5 mM</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL) 100X</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td>16 nM</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P5780</td>
			<td>4.8 &#181;M</td>
		</tr>
		<tr>
			<td>Basic FGF (FGF2), Human</td>
			<td>GoldBio</td>
			<td>1140-02-50</td>
			<td>10 ng/ml</td>
		</tr>
		<tr>
			<td>EGF, Human</td>
			<td>GoldBio</td>
			<td>1150-04-100</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Bottle-Top Filter, 150ml, 33mm, 0.22um, Pes, S, Ind</td>
			<td>Corning</td>
			<td>431160</td>
			<td>Use to filter sterlize media</td>
		</tr>
	</tbody>
</table>

flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells

세포종 (GBM)은 미국 에서만 매년 약 14000 죽음을 일으키는 원인이 되는 성인에서 가장 일반적이 고 가장 치명적인 기본 뇌 종양. 메디아 생존 진단 다음 최대한 외과 절제술, 방사선 및 temozolomide 요법으로 15 개월 미만 이다. 더 효과적인 GBM 치료법 개발에 도전 점점 더 분명 되 고 등의 탄력성이 침입, 표준 치료에 그것의 저항, 그것의 복잡 한 유전 및 분자 적응성, GBM의 모집단 정상 줄기 세포, phenotypic 유사성과 셀 본 세포종 줄기 세포 (GSCs) 라고. GSCs 종양 성장 및 진행을 위해 필요 하기 때문에 차별화에 기초를 둔 치료가 불 치의 신생에 대 한 가능한 치료 양식 적임을 나타냅니다.
다음 프로토콜 플랫폼 GSC astroglial 차별화를 홍보 하는 작은 분자의 식별 목적으로 상영 높은 처리량을 설정 하는 절차의 컬렉션을 설명 합니다. 시스템의 핵심에는 폐해 fibrillary 산 성 단백질 (GFAP) 차별화 기자-구문입니다. 다음과 같은 일반적인 절차를 포함 하는 프로토콜: (1) 설립 GSC 차별화 기자 라인; (2) 테스트/유효성 검사 GSC 자체-갱신/clonogenic 용량; 기자의 관련성 그리고 (3) 높은-용량 cytometry 마약 검사를 기반으로.
심사 플랫폼 GSCs 차별화를 홍보 하는 작은 분자를 식별 하는 간단 하 고 저렴 한 접근을 제공 합니다. 또한, FDA 승인 약품의 라이브러리의 활용 더 급속 하 게 내리거나 수 에이전트의 식별에 대 한 가능성을 보유 하고있다. 또한, 암 줄기 세포 분화를 촉진 하는 치료 대상 및 주로 더 차별화 된 암 세포를 제거 하기 위해 표시 되었습니다 현재 "표준 치료" 치료 공동 성으로 작동 하도록 예상 된다.

3 개의 독립적인 환자 파생 neurospheres 라인 Zeocin 저항 카세트 녹색 형광 단백질 (GFP) 융합에 프레임에 대 한 인코딩 lentivirus astroglial 기자와 불리고 되었고 인간의 GFAP 발기인 요소 ( 에 의해 구동 그림 1). 다음 개별 클론 96 잘 접시 (그림 2)에 잘 당 0.7 셀을 도금 하 여 고립 되었다,이 GFP (그림 3)를 표현 하는 ?...

Watch this Scientific Journal Video about Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells at JoVE.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

교류 Cytometry 기반 약물 세포종 줄기 세포의 세포 분화를 촉진 하는 작은 분자의 식별에 대 한 시스템 검사

효율적인 심사 프로토콜 astroglial 분화 세포종 줄기 세포 (GSCs)에 홍보 하는 작은 분자의 식별을 위해 제공 됩니다. 분석 결과 줄기 세포 감 별 법 기자에 의하여 향상 된 GFP (eGFP)의 식을 인간의 GFAP 발기인에 의해 구동 됩니다 기반으로 합니다.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

The overall goal of this procedure is to screen small molecule libraries for agents that promote astroglial differentiation in glioblastoma stem cells. This method can help answer key questions in the cancer stem cell field, such as what are the signaling pathways that are required for maintaining glioblastoma stem cells in the undifferentiated state. Proper execution of this technique allows for the straightforward identification of small molecules that promote glioblastoma stem cell differentiation.The implication of this technique extend toward therapy for Ehlers-Danlos glioblastoma because reducing the percentage of glioblastoma stem cells may decrease the likelihood of recurrence. This method can also be applied to setting differentiation into other cell lineages using other reporter constructs. Our report is the first to establish a high throughput approach to screen for agents that force glioma stem cells to differentiate.Working with lentivirus particles in cancer cells derived from patients can be extremely hazardous. Therefore standard procedure for working with bloodborne pathogens should always be followed. To begin, seed one million glioblastoma stem cells or GSCs in two milliliters of complete neural stem cell growth medium in a six-well plate.Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five. To the cell and virus mixture, add two microliters of polybrene such that the final concentration is eight micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5%CO2 overnight. After incubation, replace the growth medium to remove unbound virus.And centrifuge cells at 360 times G for five minutes. Then carefully aspirate medium and plate cells back in a six-well plate. And continue to incubate the cells for 24 hours.Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times G for five minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.Place the plate back in the incubator to allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pippetting up and down gently.To the dissociated cells add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Next, transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry.Record at least 10, 000 viable cells in each acquisition. In all flow cytometric analysis use parental non-transduced cells or vector-transduced nonfluorescent cells to establish baseline fluorescence. To select and expand individual subclones seed cells at a density of 0.7 cells per well in 100 microliters of neural stem cell medium in a 96-well plate.Maintain these clones in culture for 11 days. Look out for neurospheres with a diameter greater than 100 microns. We have previously established this cutoff size to retrospectively determine if the neuorsphere was originated by a self-renewing glioblastoma stem cell.This cutoff size may need optimization when working with different glioblastoma cell models. Observe the cells under a fluorescence microscope equipped with a FITC filter and mark wells that contain a single neurosphere in which approximately one 1%to 5%of the cells are GFP-positive. After marking, expand individual clones until a sufficient number of cells is available for flow cytometry analysis.Assuming the reporter clones contain 1%to 5%of GFP-positive cells, harvest 0.5 milliliters from each clone along with an equal quantity of non-transduced control cells to determine the exact percentage of GFP-positive cells. After pelleting cells at 360 times G for five minutes at room temperature, aspirate the supernatant. Add 200 microliters of dissociation reagent to each pellet and incubate the tubes in a water bath set to 37 degrees Celsius.After incubation, triturate the cells about 20 to 30 times to achieve a single cell suspension. Then add 800 microliters of HBSS to ensure minimal cell attachment or aggregation. Now transfer 200 microliters of each cell suspension into wells of a 96-multiwell plate.Finally, perform flow cytometry, recording at least 10, 000 viable cells in each acquisition. To assess the clonogenic capacity prepare single cell suspensions of differentiation reporter subclones by triturating the cells about 20 to 30 times. Then plate these cells in 100 microliters of complete neural stem cell growth medium at a density of about five to 500 cells per well in 96-multiwell plates.Maintain these cells in culture for nine to 11 days. Look for neurospheres under a light microscope. A well containing at least one single large neurosphere is considered positive.Considering parental non-transduced cells and GFP-positive lentivirus-transduced cells as controls input the total wells analyzed and the number of positive cells on the ELDA online interface. To begin screening, plate 5000 cells in 99 microliters of complete neural stem cell growth medium in a 96-well plate. Then with a 12-channel pipettor treat cells with two micromolar final concentration of diluted library compounds or with the DMSO control.Incubate the cells for 72 hours. Now add 150 microliters of cell dissociation reagent using a 12-channel pipettor in each well and incubate at 37 degrees Celsius for 20 minutes. Then gently triturate the cells in each well about 20 to 30 times using a 12-channel pipettor until a single cell suspension is achieved.Now perform flow cytometry and determine the percentage of cells expressing the GFAP:GFP reporter. First determine the cell size and viability from the forward and side scatters and gate the viable cell population. Then determine the GFP-positive cells within the viable cell population.An increase of percentage of GFP-positive cells by three standard deviations over controls is considered to be a positive hit which may be adjusted to accommodate user preference of stringency. Representative images of HSR-GBM1 GSC subclones expressing low and high levels of GFAP:GFP are shown. The subclones positive for GFAP:GFP expression were selected by flow cytometry for the patient-derived neurosphere line.Here, low GFAP:GFP indicates that less than 5%of the cells in the clone are GFP-positive, while high GFAP:GFP indicates that greater than 75%of the cells in the clone are GFP-positive. These results show that in vitro self-renewal capacity of GFAP-low subclones is higher than their GFAP-high counterparts as determined by ELDA. Therefore GH subclones are more differentiated than GL subclones.Finally, drug libraries were screened to identify agents capable to induce astroglial differentiation in GSCs. 12 drugs were found to significantly increase the percentage of cells expressing the GFAP:GFP reporter. Once mastered, this technique can be done in a few weeks if it is performed properly.Following this procedure other methods like alamarBlue assay, clonogenic assay in semisolid media, flank or intracranial xenograph injection can be performed in order to answer additional questions like the effect of selective drugs on cellular proliferation and clonogenicity in vitro and tumorigenicity in vivo. After watching this video, you should have a good understanding of how to identify compounds capable of inducing astroglial differentiation in glioblastoma stem cells.

Research

JoVE Journal

Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

라이브 셀 이미징 분석 실험 연구 세포종 뇌 종양 줄기 세포 이동과 내 습을

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

새로 형성 된 유방암 줄기 세포와 같은 세포 Apoptosis 반전 후 흐름 Cytometric 감지

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

암 줄기 세포를 표적으로 하는 반대로 신 생물 성 약 검열을 위한 생리학 환자 파생된 3D Spheroids

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

마우스 유방 상피 HC11 및 EpH4 세포의 분화

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 ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

배열된 라이브러리의 중간 처리량 스크리닝및 이중 루시퍼라제 기반 리포터를 이용한 전사 계수 조절기 식별

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

제브라피시의 1차 환자 유래 종양 이종이식의 약물 스크리닝

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

이동 및 세포 상호 작용을 연구하기 위해 패턴 뉴런에 교모 세포 종 줄기 와 같은 세포의 공동 배양

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

생체 내 비장 T 세포의 유세포 분석기에 의한 종양 유래 세포밖 소포의 면역원성 스크리닝

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

약물 내성 표현형을 조절하는 인자를 식별하기 위한 풀링된 shRNA 라이브러리 스크리닝

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

치료 유도 노인성 암 세포에서 다중 형광 노화 마커의 동시 이미징 및 유동 세포 측정 기반 검출

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...