Name: labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies
Uploaded: 2016-11-30T15:00:00
Description: Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

저자는이 연구에서 사용 된 높은 역가의 렌티 바이러스 생산을위한 파지 - EF1aL-을 DsRed-UBC-GFP-W 벡터 및 프로토콜을 제공하기 위해 보스턴 대학에서 박사 대럴 Kotton 감사합니다. 이 작품은 .NCI P30CA046934 센터 보조금은 생체 내 이미징에서 지원 DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG 번호 57-001-53 (DMC), NCI K22CA181250 (DMC)와 R01 CA140985 (CAS)에 의해 투자되었다 조직 문화는 콜로라도 AMC의 대학에서 코어.

이 프로토콜에서 동물의 사용을 필요로하는 모든 단계는 콜로라도 동물 연구 윤리위원회의 대학 (IACUC)의 지침을 따릅니다. 악기, 문화 미디어 및 기타 시약 1. 준비 <ol><li> (1 : 1), 염기성 섬유 아세포 성장 인자 (bFGF를 20 NG / ㎖), 표피 성장 인자 (EGF, 10 NG / ㎖ 조제 100 ㎖의 둘 베코 변형 이글 매체 및 한의 F-12 배지 (DMEM / F12)를 포함하는 미디어 mammosphere ) 헤파린 (4 μg의 / ㎖), 1X B27, 페니실린 (1...

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프로토콜 내에서 중요한 단계 : 시험 관내 형질 도입 동안 매체 조성물의 신중한 제어를 허용 높은 역가 렌티 바이러스 입자의 사용은 (&gt; 10 8 TU / ㎖),이 프로토콜의 성공에있어 중요한 단계이다. 고 역가 바이러스 입자의 제조를위한 다양한 방법이도 18, 19를 설명 하였지만; 이 프로토콜에서 상세히 설명한 바와 같이 제조 된 렌티 바이러?...

외과 적 절제 종양 샘플은 직접 면역 감염된 마우스에 접목되어 환자 유래의 종양 이종 이식 (PDXs)의 개발, 표준 셀 라인 이종 이식 모델에 비해 여러 가지 장점을 제공하며, 암 연구 1,2-에서 큰 진보를 나타낸다. PDXs 유지하고 제 1 통로에서 성장한 종양의 유전 적 및 생물학적 특성에 최소한의 변경으로 연속적인 통로로 확장 될 수있다; 보다 정확하게 인간 암세포 유래의 이종 3-8보다 종양 이질성을 반영한다. 이 모델은 현재 광범위하게 신약 개발 6,11의 전임상 플랫폼으로 암 생물학 4,12 연구를위한 실험 도구로, 암 치료제 9,10 개인화을위한 플랫폼으로 사용된다. 대부분의 PDXs는 이식 및 실행 가능하게 캘리퍼스를 사용하여 시간이 지남에 따라 종양의 성장을 측정 할 수있는 피하 전파됩니다. 하나전이성 질환은 PDXs를 사용하여 모델링하는 것이 더 어렵다. 특히 유방암, 다른 장기로 전이 용량 이종는 3,5,13 설명되었지만, 전이성 사이트 자발 보급의 빈도는 매우 낮다. 보고 된 경우, 전이성 부담의 식별 및 정량은 사후 표적 기관의 힘든 조직 학적 검사에 의존하고있다. 생물 발광 (루시퍼 라제, 배낭) 또는 형광을 발현하는 암세포는 (녹색 형광 단백질 GFP) 유전자 리포터는 일반적으로 뇌, 폐, 뼈, 간 심내 후 꼬리 정맥 intrafemoral 비장 주입 유방암 전이 실험 모델에서 사용 14-16. 이 모델은 기본 종양에서 보급을 우회하는 동안, 그들은 장기 친 화성 및 전이성 식민지의 메커니즘을 연구 할 가치가있다. 그러나, 기본 환자 종양 및 PDXs에서 파생 된 세포는 낮은 형질 전환 또는 전달 속도 날기를 가질 수있다g 표준 절차. 하나의 대안은 종래의 조직 배양 프로토콜을 사용하여 표시 할 수 체외 (17)에 PDX 유래 세포주를 확립하는 것이다. 이 방법은 그러나, 세포주 유도가 어렵고, 세포의 표현형을 변경할 수있는 가장 PDXs를 라벨에 적합하지 않다. 여기에서 우리는 생체 내 이미징에 적합한 렌티 바이러스 벡터와 PDX-해리 종양 세포의 전달을위한 프로토콜을 제시한다. 또, 면역 쥐에서 해리 루크-GFP 표지 PDX 세포의 심장 내 주사를 사용하여 실험 전이를 설명한다. 리포터 유전자를 발현하는 렌티 바이러스 PDX-해리 organoids의 전달을위한 기본 프로토콜은 이전에 18을 설명 하였다. 현재의 프로토콜에서는 인간 종양 세포에 대한 농축 및 100 % 전달 효율 근처 얻을뿐만 아니라 실험 유방암 검출 용 표지 PDXs 사용하기위한 추가의 방법을 설명전이. 이 프로토콜은 여러 암 다양한 발광 및 형광 마커 PDXs의 종류뿐만 아니라 유전자 발현 (관심의 유전자 즉, shRNA를 최저)의 변조를 라벨에 대해 적용 할 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>BD Biosciences</td>
			<td>354060</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>354001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco/Thermo Fisher</td>
			<td>17504-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungi-antibiotics</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>AM-105-500</td>
			<td>Digestion Buffer</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS Red Ca++/Mg++ free</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex</td>
			<td>Cultrex</td>
			<td>3433-005-01</td>
			<td>Basement Matrix Extract (BME)</td>
		</tr>
		<tr>
			<td>30C shaker</td>
			<td>NewBrunswick Scientific CO. INC</td>
			<td>Series 25 Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>70um filters</td>
			<td>Falcon</td>
			<td>7352350</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpels</td>
			<td>Fisher</td>
			<td>22079690</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine disinfectant</td>
			<td>Durvet&#160;</td>
			<td>NDC# 30798-624-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood&#160; cell lysis reagent</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuraminidase</td>
			<td>Sigma</td>
			<td>N7885-1UN</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (CD326+) microbeads*</td>
			<td>Miltenyil Biotec</td>
			<td>130-061-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lineage cell depletion Kit, mouse*</td>
			<td>Miltenyil Biotec</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator&#160;</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini MACS Magnetic Stand</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>MS or LS columns can be used, adjust to number of cells.</td>
		</tr>
		<tr>
			<td>Illumatool Tunable light system</td>
			<td>Lightools research</td>
			<td>Various</td>
			<td>For in vivo fluorescence imaging</td>
		</tr>
		<tr>
			<td>Xenogen IVIS200 imaging device</td>
			<td>Xenogen</td>
			<td>Various</td>
			<td>For in vivo luminiscence imaging</td>
		</tr>
		<tr>
			<td>Human Cytokeratin Clone MNF116 Monoclonal antibody</td>
			<td>DAKO</td>
			<td>M0821</td>
			<td>Pan-cytokeratin&#160;</td>
		</tr>
		<tr>
			<td>Epidermal Growth factor receptor antibody</td>
			<td>Cell signaling</td>
			<td>4267S</td>
			<td>EGFR</td>
		</tr>
	</tbody>
</table>

labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

이 방법은 높은 역가 렌티 바이러스 벡터 pSIH1-H1-copGFP-T2A-순수하지 않아 및 파지 - EF1aL - 루시 페라 - UBC-GFP-W를 사용하여 PDX-해리 유방암 세포의 전달에 대해 설명합니다. 이 벡터는, 감염 (그림 1a) 후 빠르면 24 시간을 시험관 내에서 전달의 효율성을 추정 허용 형광 마커를 표현한다. 가장 PDXs 들어, GFP 발현 세포 응집체의 형성은 일반적으로 관찰되는이 ?...

Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

우리는 녹색 형광 단백질과 루시 페라 제 기자를 발현하는 렌티 바이러스 입자 환자 유래의 이종 이식 (PDXs)의 안정적인 표시하는 방법을 설명합니다. 이 방법은 주 사이트 PDXs의 성장을 추적뿐만 아니라, 생체 내 이미징 시스템에서 사용 자발 실험 전이 검출을 허용한다.

종양의 성장과 전이 연구 추적 가능한 기자와 유방암 환자 유래의 이종 이식의 라벨링

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

The overall goal of this transduction protocol is to stably introduce reporter genes in patient-derived xenografts, or PDXs, to facilitate their use in experimental and spontaneous metastasis models. This method can help answer key questions in cancer research, such as the extent of excutaneous metastasis from patient-derived xenografts. The main advantage of this technique is that labeled PDX can be track, using in vivo imaging, facilitating the assessment of organized, specific metastasis without the extensive histological analysis post-mortem.Demonstrating this protocol will be Jessica Finlay-Schultz, a post-doc, who routinely applies this protocol for the label of breast cancer patient-derived xenografts. Using aseptic technique, begin by using a scalpel to cut the skin around the tumor. Then, pull the skin with forceps and use a clean scalpel to separate the skin from the tumor.When the tumor is completely exposed, use a clean set of forceps and scalpel to transfer the tissue into five milliliters of wash medium on ice. Next, discard the medium and wash the tumor two times with 10 milliliters of HBSS, supplemented with HEPES. Place the rinsed tumor in a sterile, pre-weighed 60 centimeter tissue culture plate, and weigh the plate to estimate the tumor mass.Using a scalpel, cut the tumor in half. Then cut a three millimeter disc from one of the halves, and place the disc in 10%formalin for 24 hours. Add 200 microliters of HBSS with HEPES to the rest of the tumor, and use forceps and a clean scalpel to mince the tissue into the smallest possible pieces.Transfer the fragments into a steril, 50 milliliter conical tube, and add at least one milliliter of digestion buffer, supplemented with antimicotics and antibiotics per 100 milligrams of tumor. After three hours at 30 degrees Celsius, and 125 to 200 rotation per minute, add 35 milliliters of wash medium to stop the digestion, and filter the resulting tissue slurry through a 70 micron nylon mesh into a new 50 milliliter conical tube, to remove any undigested tissue. Collect the cells by centrifugation, and re-suspend the pellet in one milliliter of red blood cell lysis buffer, for five minutes at room temperature.At the end of the lysis, restore the tonicity with 10 milliliters of wash buffer, and strain the cells through a 40 micron filter to remove any clumps. Centrifuge the cells again, and re-suspend the pellet in five milliliters of wash buffer on ice. Then count the number of viable cells by trypan blue exclusion.To enrich for the human epithelial cancer cells using lineage plus Mel cell depletion, transfer up to one times ten to the seventh cells into a new five milliliter polypropylene tube, and add two milliliters of epithelial enrichment buffer to the cells. Collect the cells by centrifugation, and use a pipette to completely remove the supernatant. Next, re-suspend the pellet in 40 microliters of ice cold epithelial enrichment buffer, and add 10 microliters of biotin antibody cocktail per one times ten to the seventh cells.Mix by gentle pipetting, and incubate the cell solution for 10 minutes at four degrees Celsius. At the end of incubation, mix the cells with 30 microliters of epithelial enrichment buffer per one times ten to the seventh cells. Followed by the addition of 20 microliters of antibiotin microbeads with gentle mixing.After 15 minutes at four degrees Celsius, wash the cells in three milliliters of cold epithelial enrichment buffer, and carefully aspirate the supernatant. Re-suspend the pellet in 500 microliters of epithelial enrichment buffer on ice, and place a column in a magnetic separator. Rinse the column with 0.5 milliliters of epithelial enrichment buffer.And place a new polypropylene collection tube under the column. Then slowly add the labeled cells over the column. Collecting the enriched, unlabeled, lineage negative, tumor cell fraction affluent, followed by three column rinses with 500 microliters of fresh epithelial enrichment buffer.To transduce the tumor cells, centrifuge the isolated cells for re-suspension in two milliliters of mammosphere medium. After counting, centrifuge the cells again, and re-suspend the pellet in 2 milliliters of mammosphere medium, supplemented with 20 microliters of polybrene. Next, plate two times ten to the fifth viable tumor cells per well in an ultra low adherence six well tissue culture plate, and add lentiviral particles to the cells at a multiplicity of infection of 10.Swirl the plate to mix the virus with the cells. And incubate the co-cultures at 37 degrees Celsius and 5%carbon dioxide for up to 96 hours. Adding 500 microliters of fresh mammosphere medium after the first 24 hours.When at least 10%of the cells are GFP positive, transfer the transduce cells from at least one well into a 15 milliliter conical tube on ice, and wash the well with one milliliter of mammosphere medium. Pool the wash with the cells, and centrifuge the tumor cells in 10 milliliters of HBSS plus HEPES. Finally, re-suspend the cells in 50 microliters of basement matrix extract on ice.And load the embedded cells into a 0.5 milliliter insulin syringe for re-implantation. Some high titer vial vectors express a fluorescent marker that allows estimation of the transduction efficiency in vitro, as early as 24 hours after infection. For most PDXs, the GFP expression will be delayed up to 72 hours after infection.At which point, the formation of cell aggregates is commonly observed. After extracellular matrix suspension, and re-implantation, the labeled tumor cells regenerate tumors that can be tracked using bioluminescence or fluorescence. A successfully labeled PDX will be nearly 100%GFP positive at the first generation post transduction, and will remain so in subsequent passages.However, some PDX's will exhibit a patchy distribution of GFP positive cells, indicating a suboptimal in vitro transduction. Immunohistochemical tumor tissue staining reveals that the expression of critical markers, such as epidermal growth factor receptor, and pan cytokeratins, are conserved even after several passages and re-implantations. Experimental models of metastasis provide an alternative to studying organ tropism and organ colonization steps in the metastatic cascade.Indeed, a transduced PDX injected at 2.5 times ten to the fifth cells per mouse, forms metastasis in the liver that are easily identified with luciferase imaging in vivo. And in the lungs that can be visualized by fluorescence microscopy ex vivo. Once mastered, the dissociation and labeling of the PDXs takes about 4 hours.The actual expansion of the tumor can take 8 to 12 weeks depending on the tumor, and system used. When attempting this procedure it's important to use a high titer virus with a reporter gene, so you can monitor both transfection efficiency and tumor growth in vivo. Following this procedure, other methods such as in vivo metastasis studies can be performed to answer other questions on tumor progression, metastasis, and response to treatments.This technique paves the way for breast cancer researchers to explore multiple aspects of tumor progression, using novel PDX models that better represent tumor heterogeneity. After watching this video, you should have a good understanding of how to label dissociated tumor cells with high titer lentiviral particles. Which will allow you to both trace tumor cells in vivo as well as potentially manipulate the expression of genes of interest.Don't forget that working with lentiviral particles can be extremely hazardous, and you should always take proper precautions when performing this procedure.

Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

종양의 전이에 저산소증의 테스트 효과에 대한 생체 내 모델에서

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

연구

암 연구학

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

육종 환자 유래 이종이식에서 종양 을 발하는 세포의 격리 및 특성화

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

인간 비뇨기과 세포 암 및 대장암 종양 성장 및 자발적 전이를 위한 환자 유래 직교 이종이식 모델

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

하류 분자 분석 및 전이성 전위 평가를 위한 순환 종양 세포의 미세 조작

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

구조적 DNA 변경 분석 및 환자 유래 제노이식을 사용하여 암에 있는 표적으로 한 치료 시험

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Perfused 마이크로 유체 플랫폼에서 환자 유래 제노이식의 Ex vivo 3D 하이드로겔 배양에 대한 향상된 생존력

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

중추신경계 전이의 환자 유래 이종이식 모델의 확립 및 활용

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

제브라피시 애벌레 제노이식 및 종양 행동 분석의 생성

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

환자 유래 종양 오르가노이드를 사용하여 시험관 분석에서 높은 처리량

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...