This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

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The authors declare that they have no competing financial interest.

암줄 기세포는 종양의 대부분이 특정 세포 부분 집합의 식별을 허용하는 여러 속성이 있습니다. 이러한 표현식, ATP 결합 카세트 다제 유출 수송차 28, 32, 33, 또는 ALDH 32 해독 효소의 발현의 상향 조절을위한의 과발현을위한 화학 요법 제를 세포 독성 취득 된 저항 이러한 특성에 기초 된 CSC를 분리 예컨대 CD133, CD44, CD34, CD90, 및 기타 30, 34, 35, 41, 여러 가지의 방법으로서, 특정 표면 마커의 42-44를 개발되었다. 이러한 기술 중 하나는 비 - 부착 조건 하에서 성장 된 CSC의 용량에 기초한 구 형성 분석이다. 구체를 형성하는 조직 줄기 세포 및 CSC 추가의 능력은 제 레이놀즈 등. (37)에 의해 신경줄 기세포의 식별에 관한 연구에서 설명 하였다. 그 후, 깁스 등. (38) 우리를이러한 연구 에디션은 골 육종에서, 특히, 고형 종양에서 암줄 기세포를 분리 시작합니다. 우리는 깁스 등의 알에 도시 된 구 형성 분석 방법을 사용 할 수 있습니다. 기존의 OS 생검에서 얻은 OSA 세포주에서 암줄 기세포를 분리하기로 결정했습니다. 우리는 원래의 방법이 분석의 결과를 개선하기위한 다른 암세포에 대한 재현성을 용이하게하도록 적응. 구 형성 검정의 설립을 참조하여, 우리는 40,000 세포를 도금하는 것은 / 웰의 분석의 시작 부분에서 분리 세포를 유지하는 것이 좋다 것을 확인 하였다. 이 트릭은 구 식민지가 세포 응집하지 않은 부착 조건에서 성장하고 구형 콜로니를 형성하는 하나의 CSC의 특정 독점 용량에서 발생 가능성을 방지하는 것이 매우 중요합니다. 이 기능은이 분석의 특히 중요한 포인트입니다. 또한 구 형성의 양호한 비율을 구하는 것을 증명원래의 방법에 기재된 바와 같이 매일 성장 인자의 분취 량을 매 3 일 아닌 새로 고침하기에 충분하다. 본 연구에서는, 우리는 또한 설립 광범위하게 비 접착 조건에서 배양 할 때 형성되는 구형 콜로니를 분리하기위한 좋은 방법을 설명했다. 이 웰을 손상시키지 않고 각각 형성 가능한 분야의 많은을 분리하려고하는 것이 매우 중요하기 때문에이 단계는이 분석에서 중요하다. 단지 구체 아니라 분석 기간 동안 현탁액에 남아 있었던 단일 세포를 분리하는 것이 중요하다. 이 중요한 점을 극복하기 위해, 우리는 위의 그림과 같이, CSC 격리를위한 좋은 결과를 준, 특정 분리 방법을 개발했습니다. 분명히이 형성하는 모든 분야가 복구 될 수없는 가능성이 있지만, 손실 비율이 매우 낮다. 실제로, 우리는 그들이 결정 (커질 후 구를 분리하여 40 ㎛의 기공 필터를 사용할 수있을에드 약 100-200 세포). 이 격리 영역 형성을 정지하지만, 단 전지의 메틸 잔기의 일부가 가장 작은 구체를 여과 할 수있다. 프로토콜에 기재 한 바와 같이 제거는 철저한 여과에 의해 수행된다. 또한, 가장 작은 구형 콜로니 그에 따른 손실이 40 ㎛의 메쉬를 통해 큰 구형 콜로니의 선택은 하나의 구형 콜로니를 형성 할 수있는 높은 용량과 더 stemness로 된 CSC를 선택할 수 있습니다. 이러한 모든 수정은 분석 향상과 이해 ​​구 형성 검정의 원래있어서 가장 중요한 단계를 재현 된 CSC 공부 연구자 있도록 하였다. 분리 암줄 기세포에 대한 시험 관내 방법에 관한 연구 중 본 연구에서 암줄 기세포를 분리이 적응 구 형성 분석은 좋은 방법이 될 수있는 방법을 보여 목표OSA 세포주. 일본어 방법 및 상세한 분리 기술에 대한 적응은 효능 향상을 설명했다. 단시간에, CSC 추가의 상당수가 얻어 질 수 있고 여러 가지 실험에 사용 하였다. 따라서, 급속 OS-CSC 추가 특징 이중 스템 형 표현형을 연구하기 위해, 특히, 줄기 등의 표현형을 확인하는 것이 가능하다. 따라서,이 수정 된 분석은 암줄 기세포를 분리 및 생물학 연구를위한 좋은 방법이 될 수 있습니다. 미래에,이 방법은 추가로 적응도 희귀 고형 종양의 생검에 의해 얻어지는 다른 유한 암 세포주에서 암줄 기세포를 분리하는데 사용될 수있다. 이러한 OS 희귀 고형 종양에서 CSC 추가 분리 가능성뿐만 아니라, 특정 암에 대한 연구의 개선을 허용하지만, 또한 분리를위한 더 좋은 방법을 개발하는 암의 다양한 유형의 연구 및 생물학의 앞으로의 연구를 확장 이 중요한 세포의 부분 집합. 따라서, 우리와 같은본 연구에서 수행 한, 아마에 대한 책임이 특정 세포의 부분 집합에 관한 매우 구체적인 항암 치료, 분자 표적을 발견하고 개발의 최종 목표로, CSC 생물학의 연구를 통해 CSC 분리의 방법을 개선하는 것이 중요하다 주 종양의 유지 재발의 개발 및 여러 기관에서 전이의 기원. CSC 생물학의 연구는 항암 치료 후 생존율이 매우 가난한 유지하는 등의 OS와 같은 암의 치료에 예리한 될 수 치료법을 찾는 것이 중요합니다.

육종은 배아 중배엽 (1)에서 주로 발생하는 드문 악성 결합 조직 종양의 이종 그룹입니다. 다른 유형은 골 육종 및 연부 조직 육종을 포함한다. 뼈 육종은 비교적 드문 차 종양의 그룹, 골육종 (OS)를 포함한 여러 아형,로 구성되어 있습니다. OS, 뼈의 일반적인 기본 종양 중 하나는, 광범위한 임상 적, 조직 학적 및 분자 이질성 (2, 3)을 나타내는 중간 엽 악성 종양이다. 불행히도, OS 5, 어린이 및 청년 4에서 주로 발생하며 60 %를 차지 어린 시절 뼈 육종의 일반적인 조직 학적 아형은 6, 7. OS는 일반적으로 영향을 빠른 뼈의 성장을 특징으로 골격 영역 (예를 들면, 긴 뼈의 골 간단). 또한 수질 또는 중앙 OS라는 OS, 기존 OS의 조직 학적으로 서로 다른 아형 중 악성 종양의 높은 학년과 할당량 샤가80 % 8 재. 이 80 %는 60 % 종래 골아 OS 10 % chondroblastic OS, 및 10 % 섬유 아세포 OS 6, 8-10로 구성되어있다. 다른 OS 서브 타입은 역 형성, telangiectatic, 거대 세포가 풍부한 작은 세포 OS를 포함한다. 결합 수술 OS 관리 화학 요법의 진보에도 불구하고, 결과는 전이 11,12없이 환자 65-70%의 장기 생존율 불량 남아있다. 먼 재발 자주 전이로, 또는 덜 빈번 폐 전이 발생할 먼 뼈와 지방 재발 13. 전이는 종종 기존 치료에 저항. 이 저항은 10 년 무병 생존율 진단 14, 15, 전이성 질환을 가진 환자의 약 30 %가되는 이유이다. 정상 조직과 같이, 암 조직은 세포 유형의 이종의 집합으로 구성된다. 종양 내 세포 발달의 각 단계에 대응하는 것으로 보인다. 상관 없음 내ormal 조직 따라서 조직 항상성을위한 전구 세포 및 성숙한 세포를 제공 selfrenew 할 수있는 능력 세포의 서브 모집단을 상주. 마찬가지로, 암의 증식과 종양 전위 상이한 정도와 개발의 다른 단계에서 세포의 유사한 이종 집단으로 구성된다. 이러한 암 세포의 서브 세트는 암 줄기 세포 (CSC 추가)를, 종양 부피 (16)를 구성하는 다른 세포 계통을 따라서, 종양의 악성 가능성 selfrenew 및 유지 배타적 능력 세포 selfsustaining 생성 리저버를 구성 되나. 1990 년대에, 급성 골수성 백혈병에 대한 연구는 CSC의 소집단 (17), (18)의 존재에 대한 최초의 강력한 증거를 제공했다. 암줄 기세포는 이후함으로써 암 연구에서 가장 연구 주제 중 하나가되고, 고형 종양 (19)의 큰 숫자에서 고립되었다. 암줄 기세포는 실제로 정상 만드는 유전자의 돌연변이에 의해 정상 줄기 세포에서 발생할 수20-23 암 줄기 세포. 미세 환경과 여러 변형 돌연변이 및 상호 작용은 암줄 기세포를 예표 selfrenewal 용량과 불멸을 획득 ​​건강 전구 세포와 성숙 세포에 기여할 수있다. 이러한 변화에 대한 여러 가지 가설이있다. 건강 선조 건강한 성숙 세포 및 암세포 selfrenewal 관련 유전자 24-28을 활성화하여 줄기 같은 표현형을 획득하는 세포를 줄기 탈분화있다. 몇 가지 최근의 연구에도 불구하고, 암줄 기세포의 기원은 아직 발견해야합니다. 암줄 기세포의 특별한 특징은 능력이 다른 약물과 결합 된 수술과 화학 요법으로 구성 다중 치료 접근 방식을, 저항하는 것입니다. 최근의 연구는 암줄 기세포는 화학 요법 제를 세포 독성에 내성을 획득 할 수 있다는 것을 보여 주었다. 이 저항에 대한 가능한 설명은 ATP 바인딩 카세트 (ABC) 다중 약물 수송의 과발현을 포함한다 (즉,MDR1 및 BCRP1) 알데히드 데 하이드로게나 제 1 (ALDH1) 및 / 또는 세포주기 동력학 30-33 변화로서 항암제 대사 효소의 과발현. 지금까지 설명 된 모든 이러한 개념의 직접적인 결과는 CSC의 하위 집단이 완전히 제거 된 경우에만 단 한 번의 CSC가 살아 경우 국소 재발이나 원격 전이가 발생할 수 있습니다 동안 암 치료가 효과적 일 것입니다. 이 환자 나중에 재발 많은 치료 초기에 효과적인 것으로 보인다 이유에 대한 가능한 설명을 제공하지만, 때문에 인간의 육종 (34), 특히 OS (35), 또는 다른 뼈와 연부 조직 암에서의 암줄 기세포의 발견은 큰 임상 적 중요성을 가지고있다. 따라서, 기존의 OS에 대한 미래의 전투에 대한 희망이 하위의 분자 특성에 OS-암줄 기세포 감사에 관한 혁신적인 약물의 개발에 따라 새로운 특정 표적으로 한 치료를 찾을 수 있습니다인구와 CSC 생물학의 연구에. 1992 년 줄기 세포의 일부는 성숙한 포유 동물의 뇌에 존재 여부를 조사 하였다 레이놀즈와 동료, 줄기 형 셀 (36), (37)로 의심되는 세포를 분리하는 방법을 개발 하였다.이 방법의 특정 기능에 기초 비 접착 조건 하에서 성장 될 때이 세포 구형 콜로니를 형성한다. 비슷한 기술이 골 육종 (38)의 줄기와 같은 세포의 모집단을 연구하기 위해 2005 년에 깁스와 동료에 의해 사용되었다. 분리 종래 OS의 종류의 일차 세포 배양 물로부터 OS-CSC 추가 특성화하기 위해 OS 세포주에이 기술을 적용하기로 결정했다. 여기서는 종래의 OS의 생검 인간 유래의 유한 차 세포주로부터 OS-CSC 추가를 분리하는데 사용될 수있다 &quot;sarcosphere 분석&quot;이라 구 형성 분석이 적응 방법을 서술. 우리는 또한 모든 기술 유를 설명이 분석에 의해 단리 세포주의 줄기 등 CSC 표현형 확인 나오지 : 그리고 암줄 기세포의 마커 인 CD133 유전자의) 다 능성 배아 줄기 세포 (는 ESC를 특성화하는 유전자의 발현의 1) 평가하는 단계; 2) 집락 형성 단위 (CFU) 분석; 3) 적당한 분화 조건에서 조골 세포 및 지방 세포로 분화하도록 이들 세포의 기능 평가; 면역 형광 염색 및 유세포 분석에 의한 중간 엽 줄기 세포 표면 마커 (MSC들) (즉, CD44, CD105 및 STRO-1) ~ 4) 연구; 이들 세포의 활성 ALDH 5) 평가.

establishment of cancer stem cell cultures from human conventional osteosarcoma

골육종 (OS)에 대한 치료에 현재 향상 암 환자의 생명을 연장했지만 전이가 발생했을 때, 5 년 생존율은 여전히 ​​좋지. 암 줄기 세포 (CSC) 이론은 종양을 유지하고 약제 요법에 저항 할 수있는 능력을 포함한 줄기 같은 특성을 갖는 종양 내 종양 세포의 서브 세트가 있음을 보유하고있다. 따라서, OS의 생물학 및 병인에 대한 이해는 특정 서브 세트를 박멸 및 환자군 질병률 및 사망률을 감소시키는 치료 표적의 개발을 진행시키기 위해 필요하다. , 암줄 기세포를 분리 암줄 기세포의 세포 배양을 수립, 그들의 생물학을 공부하는 것은 OS 생물학 및 병인에 대한 우리의 이해를 향상시키는 중요한 단계입니다. OS의 생검에서 인간 유래 된 CSC-OS의 설립 nonadhere 하에서 3 차원 줄기 세포 배양 물을 만들 수있는 능력을 포함하여, 여러 가지 방법을 사용 가능하게 된NT 조건. 이러한 조건에서, CSC 추가 딸 줄기 세포에 의해 형성된 콜로니 구형 플로팅을 만들 수있다; 이 식민지는 &quot;세포 분야&quot;라고되어있다. 여기서는 OS 생검에서 얻어진 종래의 OS의 일차 세포 배양 물로부터 CSC 배양을 설정하는 방법을 설명한다. 우리는 명확하게 암줄 기세포를 분리하고 특성화하는 데 필요한 여러 구절을 설명합니다.

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

뼈 육종 암 줄기 세포의 존재 (CSC 추가)는 최근에 발병에 연결되어있다. 이 글에서, 우리는 비 접착 조건에서 성장하는 암줄 기세포의 능력을 사용하여 기존의 골육종 (OS)의 인간 생체 검사에서 얻은 기본 세포 배양에서 암줄 기세포의 분리를 제시한다.

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

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Cancer Research

18.9K 조회수.  University of Florence.  뼈 육종 암 줄기 세포의 존재 (CSC 추가)는 최근에 발병에 연결되어있다. 이 글에서, 우리는 비 접착 조건에서 성장하는 암줄 기세포의 능력을 사용하여 기존의 골육종 (OS)의 인간 생체 검사에서 얻은 기본 세포 배양에서 암줄 기세포의 분리를 제시한다. 

비디오: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

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

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

종양 및 중간 엽 줄기 세포 사이의 세포 외 기 중재 통신을 정의 하는 다리의 전 임상 마우스 모델

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

연구

암 연구학

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li Fraumeni 증후군 환자에서 파생 된 유도 만능 줄기 세포를 사용 하 여 다리를 모델링

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

등쪽 뿌리 신경절 축삭-올리고젠드로시트 공동 배양에 대한 인간 신경교종 세포 이동의 실시간 모니터링

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

위암 환자 유래 이종이식 모델 및 1차 세포주 구축

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

부인과 및 유방암 종양에서 암 줄기 세포 구체 획득

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3차원 스페로이드 배양에서 줄기와 같은 세포의 분리

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

아데노 관련 바이러스-Cas9 시스템을 사용하여 유전자 조작 된 뮤린 머리와 목 암 세포라인의 시험관 내 설립

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

대장암세포의 3차원 스페로이드 배양에서 프로바이오틱스의 세포없는 슈퍼네탄을 사용하여 세포 사멸 평가

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

세포 및 조직 샘플에서 인간 유방암 줄기 세포의 분리 및 기능 평가

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

감마 델타(γδ) T 세포의 확장 및 농축

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...