이 작품은 힐러리 공예, 선생님 더글러스 마이어스 특별한 언급과 제국 BRC, 건강 연구를위한 국립 연구소 및 조치에 대하여 암에 의해 지원됩니다.

절차는 다음과 윤리적 임페리얼 칼리지, 런던에 의해 승인되었습니다. 인간 유방암 세포주에서 차 Mammospheres의 1 세대 참고 : 멸균 문화 후드에서 다음 단계를 수행하십시오. <ol><li> Mammosphere 미디어 2 mM의 L- 글루타민이 보충 된 DMEM / F12, 100 U / ㎖ 페니실린, 100 U / ml의 스트렙토 마이신을 함유하는 준비한다. 10 NG / ml의 재조합 인간 염기성 섬유 아세포 성장 인자 (bFGF; R &amp; D 시스템) 및 1X B27 보충제 20 NG / ml의 재조합 인간 상피 성장 인자 (시그마 EGF)을 첨가하여 사용 직전에 완료 매체를 준비한다. </li><li> (또는 원하는 유방암 세포주 70-80 % 컨 플루 언스 (confluence)에) 부착 MCF-7 또는 MDA-MB-231 세포를 함유하는 플라스크 대기음 매체는 1X PBS에 두 번 세척하고, 세포를 Trypsinize. </li><li> 실온에서 5 분 동안 원심 분리기 (200) XG 세포. mammosphere 미디어의 1-5 ml의 디 캔트 뜨는에 resuspend 세포. 피펫40 μm의 셀 스트레이너 캡 필터 상하 필요한 사용하는 경우, 단일 세포 현탁액을 얻었다. 단일 세포 현탁액 (그렇지 않은 경우, 3 회에 세포 현탁액을 주사기 25 G 바늘을 사용하여) 형성하고 있도록 혈구를 사용한다. </li><li> FACS를 통해 필요한 분리 된 CD44 + CD24- 세포의 일부는 항 CD24 피코 에리 트린 (PE) 및 항 CD44-형광 이소 티오 시아 네이트 (FITC) 단일 클론 항체 (9)를 사용하는 경우. 또한, 자기 활성화 된 셀 정렬 안티 CD44 및 제조업체의 지침에 따라 항 CD24 - 바이오틴 결합 된 마이크로 비즈 (MACS) 시스템을 사용하는 인구를 분리. LD 열을 사용하여 LS 열을 사용하여 긍정적 인 선택, 음의 선택을 수행하고 17 유동 세포 계측법에 의해 모든 격리 세포의 표현형을 확인합니다. </li><li> 트리 판 블루를 사용하여 1 mL 당 생존 세포의 수를 계산합니다. 주 : 세포 생존율에 hemocy 그리드 내의 셀의 총 개수로 나누어 생존 세포의 수로서 계산된다tometer. 트리 판 블루를 차지 세포가 아닌 실행 가능한 것으로 간주됩니다. 다음 절차를 정확하게 생존 세포의 비율을 결정하는데 사용된다. <ol><li> PBS에서 트리 판 블루의 0.4 % 용액을 준비합니다. 세포의 1 ml의 트리 판 블루 원액 0.1 ML을 추가합니다. 혈구를 넣고 낮은 배율 현미경으로 즉시 검사합니다. 총 셀의 개수와 블루 염색 세포의 수를 계산. 가능한 세포 = [1.00 - (파란색 셀의 수 ÷ 총 세포 수)] × 100. </li><li> 배양 1 ㎖ 당 생존 세포의 수를 계산하는 아래의 식을 사용한다. 희석 배수를 보정해야합니다. 104 × 1.1 = 세포 / ㎖ 문화 × 가능한 세포의 수 </li></ol></li><li> 6- 웰 초저 부착 플레이트의 각 웰에 전체 mammosphere 미디어 2 ㎖로 세포의 사전 결정된 양을 재현 탁. 파종 밀도는 일반적으로 500-4,000 세포 / cm 아니라 당이 세포입니다. 선택적으로 사용 싸다w 부착 24 웰 플레이트는 매체의 0.5 ml의 동일한 밀도로 세포를 파종한다. 참고 : 우리는 관심의 각 세포주에 대한 파종 밀도와 문화의 시간을 최적화하는 것이 좋습니다. </li><li> 특히 제 오일의 성장 단계에서, 플레이트를 방해하지 않고 (세포주 및 mammospheres의 크기에 따라) 5-10일 37 ° C, 5 % CO 2에서 초저 부착 6 웰 플레이트를 인큐베이션 . </li></ol> 인간의 유방암 임상 샘플에서 차 Mammospheres의 2 세대 주 : 인간 유방암 조직 유방 종양의 제거를위한 수술을받은 환자로부터 얻을 수있다. 100 U / ml의 페니실린 100 U / ml의 스트렙토 마이신을 포함하는 배지에서 50 ml의 멸균 튜브에 최대 24 시간 동안 얼음에 저장 조직. 멸균 문화 후드에서 다음 단계를 수행합니다. <ol><li> 미디어의 작은 부피와 100mm 조직 배양 접시에 시료를 전송합니다. Remov멸균 가위, scapel 및 핀셋을 사용하여 전자 지방 조직. </li><li> DMEM / F12의 2-3 ML을 추가하고 더 큰 조각이 남아 있지 않을 때까지 살균 scapel 또는 면도날 1mm 3 조각으로 샘플을 말하다. </li><li> 목록 조직편 재현 탁 단백질 분해 효소 (3,000 U / ml의 콜라겐 및 1000 U / ㎖의 히알루로니다 아제)를 함유하는 10 ㎖의 DMEM을 프리 - 가온하고 모든 조직 파편이 소화 될 때까지 회전 진탕하에 37 ℃에서 배양한다. 일반적으로 완전한 소화은 1-3 시간 정도 걸립니다. 종양의 병리학 자에 따라 소화 시간을 조정합니다. (예를 들어 유방 선암 더 취약 짧은 소화 시간이 필요 점액 성 암종에 비해 소화가 일반적으로 어렵다). 혈구에서 20 μl의 서스펜션을 관찰하여 소화 모든 ½ 시간의 정도를 평가합니다. </li><li> 단편 후, 5 분 동안 침강 실 TEM에서 10 분 동안 200 XG에 15 ml의 원추형 폴리 프로필렌 원심 분리 튜브에 상청을 전송하도록 허용아네트. mammosphere 매체 5 ㎖ - 조심스럽게 1에 재현 탁하고 세포 상등액을 가만히 따르다. </li><li> 취득 및 단일 세포 현탁액을 도금하는 세포주를 위해 위의 설명 된 단계 1.1 ~ 1.4을 따릅니다. 필요한 경우 G (25)를 통해 세포가 세포 손상을 제한하기 위해 2 배의 최대 시린지 패스. </li></ol> 3. Mammosphere 효율 (%) 계산 성형 <ol><li> 배양 기간 후, 40 배 배율 현미경 mammospheres (이상 40 μm의) 직경을 계산합니다. 디지털 화상 (5) 임의의 광학 현미경에 디지털 카메라를 사용하여 필드 및 수집 소프트웨어를 이용 mammospheres 크기를 결정한다. 어떤 이미지 분석 소프트웨어를 사용합니다. </li><li> 다음 식을 이용 Mammosphere 형성 효율 (MFE의 %)을 계산한다 MFE (%)는 = (웰당 mammospheres의 #은) / (웰 당 세포의 접종은 #) × 100 </li></ol> 자기 갱신의 평가를위한 Mammospheres 4. 직렬과 Passaging <ol><li&gt; 피펫 15ml를 튜브에 각 웰로부터 mammospheres 함유 매체. PBS로 우물을 세척하고 수집 된 미디어에 추가 할 수 있습니다. 실온에서 10 분 동안 원심 분리기 (115) XG. </li><li> 뜨는을 취소하고 미리 예열 0.5 % 트립신 / 0.2 % EDTA의 500 μL에 펠렛을 재현 탁. 2 ~ 3 분 동안 품어. </li><li> 트립신을 중화 FCS 500 μl를 추가합니다. 5 분 동안 500 XG에 원심 분리기. 구체들을 분해 아래로 mammospheres 미디어, 피펫 최대 100 ㎕에 뜨는에 resuspend 펠렛을 취소하고. </li><li> 혈구와 세포를 세어 세포는 단일 세포 현탁액에있는 경우 결정합니다. 25 G를 통과하지 않으면 단일 세포를 얻기 위해 3 회까지 주사기. 기본 생성에 사용되는 동일한 농도에서 새로운 초저 부착 6 웰 플레이트에 세포를 시드. </li><li> 5-10일 후, 구&gt; 40 μm의 수와 이전에보고 된 공식을 사용하여 구체 형성 효율을 계산한다. </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

기본 및 보조 mammospheres의 성공적인 평가는 세포가 최소한의 구 집합으로, 단일 클론에서 양식을 mammospheres 충분히 낮은 밀도에서 도금되는에 의존합니다. 그러나 너무 낮은 밀도가 너무 적었 mammospheres 통계적 처리의 효과를 구별하기 위해 형성 할 수있다. 파종 밀도가 그들의 영역 형성의 효율에 상당히 다를 수 있기 때문에 사용 된 각각의 세포주에 대해 최적화되어야한다 (낮은 E-cadherin의 표현?...

줄기 같은 암 줄기 세포가 이끄는 종양 세포 계통의 존재는 크게 종양 이질성에 대한 우리의 이해에 추가했다. 종양의 일부 표현형 다양성 유전자 구별 클론의 클론 생장에서 발생 않지만, 실질적인 성분은 후생 유전 학적 차이에 기인하는 것으로 나타난다 : 암 세포가 특정 유전자의 활성화 또는 억제를 통해 줄기, 전구 및 분화 상태 사이 (가역적 때때로) 전환 할 수 식 프로그램 1-3. 이것은 현재 조절 성 요소를 제공하는 암, 간질 또는 면역 인접 셀로부터 측 분비와 함께 그 결과의 분비 신호와 세포에서 발현되는 유전자 발현 프로그램을 반영 셀 고유 또는 외인성 인자를 반영하고있다 같은 정도로서 microenviromental 조건 저산소증 2,4,5. 혁신적인 혈통 추적 방법이지만이들 생체 내 틈새 (6)에 추정되는 암 줄기 세포를 연구 할 수있는 능력 전진 - 8 구 - 형성 분석법 적어도 사용 분석 조건 하에서, 줄기 세포처럼 행동하도록 유방암 세포 잠재력을 추정 인기 편리한 접근 남아있다. 그것은 종종 더 mesenchymal- 상피 대응하도록 제안되어 막 마커 CD44 및 CD24 9 및 효소 ALDH (알데히드 탈수소 효소)의 활동 수준 (10), 마커의 발현에 의해, 정화 암 줄기 세포 대 향적 방법과 함께 사용 -like 암 줄기 세포 각각 11. 구 형성 방법이 제 상피 성장 인자 (EGF) (12)의 첨가 비 점착성, 무 혈청 조건에서 단일 클론으로부터 추정되는 줄기 세포의 성장을 가능 neurosphere 분석법 개발되었다 나중에 유용 정상 및 암에 적용되는 유방 조직. 뼈 선의의 줄기 세포는 생체 내에서 활성화를 선호 할 요인의 정확한 조합을 발생하지 않습니다, G0 단계에서 내립니다. mammosphere 분석 대신 유사 분열 부문 태세 이미 13을 분할하거나 세포의 성장을 할 수 있습니다. 이들 전구 세포가 아닌 세포 진정한 정지되지만, 분석에 사용 된 EGF 및 염기성 섬유 아세포 성장 인자 (bFGF)와 유사 분열 증식 세포 단계 일 수있다. 그럼에도 불구하고, 그들은 활성화 줄기 세포 - 관련 시그널링의 범위 (14) 경로를 포함하고있다. 또한, 그 형성 속도는 마우스 이종 이식편에서 2,15,16을 제한 희석 분석에서의 역가 측정들이 찍은 된 조직의 종양 발생에 관한 &lt;/ SUP&gt;. 여기서 우리는 단일 세포를 분리 및 인간 유방암 세포주 및 유방암의 임상 샘플 모두로부터 mammospheres 차를 생성하기 상세한 프로토콜을 제공한다. 우리는 또한 자기 갱신을 평가하는 주요 mammospheres의 일련 구절을 수행하는 방법, 및 구 다른 시드 밀도에서 비교를 (그림 1의 방식을 참조) 할 수 있습니다 효율성을 형성 계산하는 방법에 대해 설명합니다.

<table><tbody><tr><td>DMEM/F12</td><td>Lonza</td><td>CC-3151</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Sigma Aldrich</td><td>G8540</td><td></td></tr><tr><td>100 U/ml Penicillin &amp;amp; Streptomycin</td><td>Sigma Aldrich</td><td>P4083</td><td></td></tr><tr><td>20 ng/ml Recombinant human epidermal growth factor (EGF)</td><td>Sigma Aldrich</td><td>E9644</td><td></td></tr><tr><td>20 ng/ml Recombinant human basic fibroblast growth factor (bFGF)</td><td>R&amp;amp;D systems</td><td>233-FB-025</td><td></td></tr><tr><td>1x B27 supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Scientific</td><td>12399902</td><td></td></tr><tr><td>0.5% trypsin/0.2% EDTA</td><td>Sigma Aldrich</td><td>59418C</td><td></td></tr><tr><td>Fetal calf serum</td><td>First Link UK</td><td>02-00-850</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>93595</td><td></td></tr><tr><td>Low attachment 6-well plates</td><td>Corning</td><td>CLS3814</td><td></td></tr><tr><td>Collagenase type 1A</td><td>Sigma Aldrich</td><td>C9891</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma Aldrich</td><td>H3506</td><td></td></tr><tr><td>Sterile razor blades</td><td>Fisher Scientific</td><td>12443170</td><td></td></tr><tr><td>Sterile scalpel</td><td>Fisher Scientific</td><td>11758353</td><td></td></tr><tr><td>Sterile micro-dissecting scissors</td><td>Sigma Aldrich</td><td>S3146</td><td></td></tr></tbody></table>

mammosphere formation assay from human breast cancer tissues and cell lines

건강한 조직 많은 혈액 및 고형 악성 종양과 유사하게이 더욱 분화 된 자손을 발생주기 동안 자기 - 갱신 줄기 같은 암세포의 서브 세트로, 계층 적으로 구성하는 것으로 생각된다. 이해 및 향상된 화학 치료 및 비 - 조혈 종양 부피에 비해 전파 저항을 가질 수있다 유방암 이러한 암 줄기 세포를 표적으로는, 중요한 연구 분야가되고있다. mammosphere 분석이 또한 널리 향적 sphere-를 식별하는 능력을 위해 사용되었다 : CD44, CD24, 및 ALDH 활성 포함 마커 전향 면역 결핍 생쥐에 주입 할 때 향상된 발암 표시 세포를 분리하는 (FACS)를 정렬 형광 활성화 된 세포를 사용하여 평가할 수있다 하나의 줄기 세포와 같은 세포 클론 개발을 형성한다. 여기에서 우리는 세포 라인 또는 기본 환자 샘플, 자신의 계대 및 구 F를 추정하는 계산에서 mammospheres의 적절한 배양 방법을 간략하게 설명orming 효율 (SFE). 먼저 우리는 적절한 계획과 mammosphere 실험의 해석에 주요 고려 사항과 함정에 대해 설명합니다.

다른 샘플이나 다른 치료를 실시 그 시드 초기 세포 정상화 후 형성 mammospheres&gt; 40 μm의 수에 따라 다를 수 있습니다. 세중에서 재배 각각의 치료를위한 mammosphere 형성 효율 (MFE)를 계산합니다. 이것은 다른 시드 밀도 실험을 비교 할 수 있습니다. 데이터는 가장 이상적 양성 및 음성 대조군으로, 막대 그래프에 표시하고, 삼중 웰 걸친 표준 편차를 표시한다. 지속적으로 60-80% 합류 사이에 비 부착 ?...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

인간 유방암 조직과 세포주에서 Mammosphere 형성 분석

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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Medicine

33.2K 조회수.  Imperial College London. Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

비디오: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

마이크로어레이를 사용하여 암 세포 표현형에 대한 미세 환경적 영향을 심문

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

연구

암 연구학

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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 of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

의학

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

인간 유방 상피 세포에 있는 종양원성 변환의 시험관 내 평가

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological 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 fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

미생물 시스템을 사용하여 인간 유방 조직에서 유방암 모델링

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 ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

마우스 내유 상피 세포는 Lactogenic 차별 동안 Mammospheres를 형성

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

생물학

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

유방암 연구를 위한 잠재적인 기능적인 시험관 내 모형으로 유방 상피 및 내피 세포 스페로이드

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

뮤린 맘모스피어 문화에서 자체 갱신의 정량화

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

발생학

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

비 효소, Mammospheres의 자연 발생에 대한 사전 침습 유방 병변의 혈청이없는 조직 문화

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...