The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1. 다른 미디어와 추가 구성 요소를 준비 <ol><li> 실험에 앞서, 테스트 할 중 정상 FBS를 첨가, 숯 제거 FBS, 또는 숯을 박탈 FBS 플러스 요소와 DMEM 또는 지정된 다른 매체로 구성된 미디어를 준비합니다. 여러 구성 요소가 각각의 실험에서 테스트 할 수 있습니다. </li><li> 칭량 적절 생리적 농도 목탄 스트립 혈청 중에 용해되는 호르몬, 성장 인자, 사이토 카인 또는 희석. </li></ol> 2. 얼음에 콜라겐 매트릭스를 준비합니다 <ol><li> 200 μL의 10 배 PBS (PH 7.4), 5.4 ㎕의 1 N NaOH를 두 번 증류 H 2 O의 600 μL, 1.2 ml의 콜라겐 I : I는 얼음에 다음 멸균 여과 구성 요소를 추가하여 2.2 ㎎ / ㎖에서 매트릭스 2 ml의 콜라겐을 준비 AT (3.63 ㎎ / ㎖). </li><li> 판 할 준비가 될 때까지 얼음에 콜라겐 I 솔루션을 유지합니다. </li></ol> 3. &lt;분석에 대한 마이그레이션 / 침략 플레이트를 준비/ P&gt; <ol><li> 각 세포주가 시험 될 들어 12- 웰 인서트를 포함하는 24 개 웰 플레이트 챔버를 사용한다. 화학 유인 물질 미디어를 추가하고 실험 세트까지의 삽입을 전송하기 위해 삽입을 포함하지 않는 추가 12 우물을 사용합니다. 주 : 세포주 여기 용도 폴리에틸렌 텔레 프탈레이트 (PET) 멤브레인 및 8 ㎛의 공경 플레이트 콜라겐 매트릭스가 최적이다. 단, 프리 코트 번호판 마트 리겔 매트릭스는 세포주가 조사되고 감소에 따른 기공 크기로 치환 할 수있다. </li><li> 분명히 (제어, 비 침습적 세포주에 대한 FBS 마이그레이션, CS-FBS 마이그레이션, FBS 침공하고, CS-FBS의 침략뿐만 아니라 FBS 이주) 분석되는 조건 당 3 우물을 사용하여 판 레이블을 지정합니다. 콜라겐이나 마트 리겔 매트릭스를 통해 다음 막에 구멍을 통해 이동하여 PET 막에 구멍을 통해 이동하고, 분석의 침략에 의해 분석 마이그레이션. 각 셀 조건에 대해 다른 색 마커를 사용하여도금 프로세스 ID. </li></ol> 4. 침략 매트릭스를 분배 <ol><li> 조심스럽게 인서트로 콜라겐 기질 용액 75 μL 침윤 분석에 사용되는 피펫. 거품을 피하기 위해주의해야합니다. 표면에 반전 된 피펫 팁을 적용함으로써 기포를 분산. </li><li> 고화 겔 있도록 30 분 동안 37 ° C, 5 % CO 2 인큐베이터에 콜라겐 코팅 된 플레이트 인서트를 전송합니다. </li></ol> 5. 접시 막 또는 침략 매트릭스 위에 셀 <ol><li> 한편,를 Trypsinize 세포와 10 % FBS와 미디어를 추가 할 수 있습니다. 스핀 세포 테이블 상단 원심 분리기에 5 분 200 XG에 혈청이없는 배지에서 배를 씻어. </li><li> 무 혈청 배지에서 재현 탁. 혈구 또는 자동 슬라이드 카운터 세포를 계산합니다. 5 × 104 세포 / ml의 최종 농도를 혈청이없는 배지를 추가한다. </li><li> 콜라겐 매트릭스가 (30 분 후) 고체화되면, 매체로의 700 μl를 추가하거나2 % 정의 된 FBS 또는 각 웰에 FBS를 숯 - 제거. 접시 당 12 인서트 : <ul><li> 3 인서트는 미디어와 콜라겐과 우물이 + 2 % FBS </li><li> 3 인서트는 미디어와 콜라겐과 우물이 + 2 % CS-FBS </li><li> 3 삽입 미디어 + 2 % FBS와의 콜라겐과 우물이 없습니다 </li><li> 3 인서트는 미디어와의 콜라겐과 우물이 없습니다 + 2 % CS-FBS </li></ul><ol><li> 테스트되는 요소의 수와 각 조건에 대응 없음 콜라겐 따라 추가 제어 플레이트를 사용한다. </li></ol></li><li> 모든 마이그레이션 및 침략 삽입에 삽입 당 500 ㎕의 세포를 도금, 5 × 10 4 세포 / ml의 삽입에 세포 현탁액을 추가합니다. </li><li> 37 ° C에서 22 시간 동안 세포를 품어. </li></ol> 6. 마이그레이션 및 침입하는 세포의 수를 정량화 <ol><li> 별도의 우물에서 메탄올 정착, 에오신 및 hemotoxylin를 사용하여 우물의 설정 염색. </li><li> 각 우물에서 세포와 매트릭스를 제거하기 위해 면봉을 사용합니다.각 웰에 대한 두 번째 면봉 응용 프로그램과 함께 Rrepeat. </li><li> 집게로, 연속 3 각 솔루션에 각 삽입 1 초 동안 5 번 찍어. </li><li> 인서트 / N을 O를 건조하도록 허용합니다. </li><li> 어느 I) 가장자리 주위에 조심스럽게 절단, 메스 필터를 제거하고 커버 슬립과 침수 오일 슬라이드에 장착, 또는 ⅱ) 인서트 O / N 반전 건조 현미경 직접 삽입을 사용할 수 있습니다. </li><li> 다음 날, 20 배의 목적으로 현미경 슬라이드 또는 삽입을 확인하고 필터의 다른 지역에서 5 이미지를 가져 가라. 일관성을 개선하기 위해 4 외부 필드와 하나의 중심을. </li><li> ImageJ에 소프트웨어를 사용하여 모든 조건에 대한 세포를 계산하고 아래 공식에 적용됩니다. </li><li> 다음과 같이 %의 침공을 결정 : 나는 = 삽입 콜라겐을 통해 침입 세포의 # 평균 제어 삽입을 통해 마이그레이션 세포의 # = B 평균 %의 침략 = (A / B) * 100 </li><li> 다음과 같이 2 % FBS의 침략 지수를 결정 세포의 %의 침공 (2 % FBS에서) 분석되고 = C % (2 % FBS)의 제어 비 침습적 세포의 침윤 = D 내습 지수 (FBS) = (c / d) </li><li> 다음과 같이 2 % CS-FBS의 침략 지수를 결정 세포의 %의 침공 = E (2 % CS-FBS에서) 분석되고 %의 (2 % CS-FBS)에서 제어 비 침습적 세포의 침윤 = F 내습 지수 (CS-FBS) = (E / F) </li></ol> 7. 실험 프로토콜 +는 X N 여러 요인이 결합하여 숯불 제거 FBS에 FBS를 숯불 제거 비교 <ol><li> &quot;X&quot;또는 구성 요소의 조합에 대해 서로 다른 구성 요소를 사용하여 필요에 따라 절차를 여러 번 반복합니다. </li><li> 마이그레이션 및 침략 효과에 대한 각 요인 &quot;X&quot;의 기여를 결정하기 위해 계산을 적용합니다. </li></ol>

<ol>
	<li>Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of Cancer. , (2013).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li><li>Valastyan, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Dang, Z. C., Lowik, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+serum+factors+by+charcoal+treatment+promotes+adipogenesis+via+a+MAPK-dependent+pathway.">Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway.</a> Mol Cell Biochem. 268 (1-2), 159-167 (2005).</li><li>Yohay, D. A., Zhang, J., Thrailkill, K. M., Arthur, J. M., Quarles, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serum+in+the+developmental+expression+of+alkaline+phosphatase+in+MC3T3-E1+osteoblasts.">Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts.</a> J Cell Physiol. 158 (3), 467-475 (1994).</li><li>Zucker, S. N., Bancroft, T. A., Place, D. E., Des Soye, ., Bagati, B., Berezney, A., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+negative+Cx43+mutant+differentially+affects+tumorigenic+and+invasive+properties+in+human+metastatic+melanoma+cells.">A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells.</a> J Cell Physiol. 228 (4), 853-859 (2013).</li><li>Fox, E. M., Andrade, J., Shupnik, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+actions+of+estrogen+to+promote+proliferation:+integration+of+cytoplasmic+and+nuclear+pathways.">Novel actions of estrogen to promote proliferation: integration of cytoplasmic and nuclear pathways.</a> Steroids. 74 (7), 622-627 (2009).</li><li>Meierjohann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP13+mediates+cell+cycle+progression+in+melanocytes+and+melanoma+cells:+in+vitro+studies+of+migration+and+proliferation.">MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation.</a> Molecular Cancer. 9, 201 (2010).</li><li>Smalley, K. S., Haass, N. K., Brafford, P. A., Lioni, M., Flaherty, K. T., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+must+be+targeted+to+overcome+drug+resistance+in+cell+lines+derived+from+melanoma+metastases.">Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases.</a> Mol Cancer Ther. 5 (5), 1136-1144 (2006).</li><li>Both, N. J., Vermey, M., Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+evaluation+of+various+invasion+assays+testing+colon+carcinoma+cell+lines.">A comparative evaluation of various invasion assays testing colon carcinoma cell lines.</a> Br J Cancer. 81 (6), 934-941 (1999).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis:+the+'seed+and+soil'+hypothesis+revisited.">The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.</a> Nat. Rev. Cancer. 3, 453-458 (2003).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+to+mesenchymal+transition+in+human+breast+epithelial+cells+transformed+by+17beta-estradiol.">Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol.</a> Cancer Res. 67 (23), 11147-11157 (2007).</li><li>Huang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+regulation+of+miR-21+enhances+migration+and+invasion+of+hepatoma.">Thyroid hormone regulation of miR-21 enhances migration and invasion of hepatoma.</a> Cancer Res. 73 (8), 2505-2517 (2013).</li><li>Scheel, C., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+and+epithelial-mesenchymal+transition:+concepts+and+molecular+links.">Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.</a> Semin. Cancer Biol. 22 (5-6), 396-403 (2012).</li><li>Kojima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+TGF-beta+and+stromal+cell-derived+factor-1+(SDF-1)+signaling+drives+the+evolution+of+tumor-promoting+mammary+stromal+myofibroblasts.Proc.">Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.Proc.</a> Natl Acad Sci U S A. 107 (46), 20009-20014 (2010).</li><li>Maehara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transforming+growth+factor-&#946;1+in+invasion+and+metastasis+in+gastric+carcinoma.">Role of transforming growth factor-&#946;1 in invasion and metastasis in gastric carcinoma.</a> J. Clin. Oncology. 17 (2), 607-614 (1999).</li><li>Lu, Z., Jiang, G., Blume-Jensen, P., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor-induced+tumor+cell+invasion+and+metastasis+initiated+by+dephosphorylation+and+downregulation+of+focal+adhesion+kinase.">Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase.</a> Mol. Cell Biol. 21 (12), 4016-4031 (2001).</li><li>Korkaya, H., Liu, S., Wicha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+stem+cells,+cytokine+networks,+and+the+tumor+microenvironment.">Breast Cancer stem cells, cytokine networks, and the tumor microenvironment.</a> J. Clin. Invest. 121 (10), 3804-3809 (2011).</li><li>Rosen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interrogation+of+HGF-SF+with+other+cytokines+in+tumor+invasion+and+metastasis.">The interrogation of HGF-SF with other cytokines in tumor invasion and metastasis.</a> EXS. 65, 301-310 (1993).</li><li>Sanz-Moreno, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+and+JAK1+signaling+cooperate+to+control+actomyosin+contractility+in+tumor+cells+and+stroma.">ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma.</a> Cancer Cell. 20 (2), 229-245 (2011).</li><li>Fleming, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+interactions+between+primary+human+human+fibroblasts+enhance+murine+mammary+gland+humanization+in+vivo.">Paracrine interactions between primary human human fibroblasts enhance murine mammary gland humanization in vivo.</a> Breast Cancer Res. 14 (3), (2012).</li><li>Sikora, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.J.,+V.,+M.E.,+M.D.,+J.M.">M.J., V., M.E., M.D., J.M.</a> Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation.Breast Cancer Res Treat. 134 (3), 1027-1039 (2012).</li></ol>

The authors have nothing to disclose.

종양의 전이는 다단계 과정이다. 세포 기저막을 뚫고 그들이 원위부로 이송되는 림프계 또는 혈액 중 미세 혈관 내로 intravasate한다. 종양 세포를 extravasate 및 12에 정착하는 macrometastasis. 중간 엽 전이 (EMT), 종양의 침윤과 전이에 상피를 통해 진행이 스테로이드 호르몬 13, 14로 향상되었습니다, 성장은 15 ~ 18, 사이토 카인 19-21 요인. 이들 분자는 분석의 발전은 종양 ?...

신규 침윤 분석은 특정한 호르몬, 암세포의 침윤 등의 화학 유인 물질 사이토킨 및 / 또는 성장 인자의 참여를 식별하기위한 목적으로 개발되었다. 세포 외 기질 (ECM)를 통해 침투하는 종양 세포의 능력은 전이성 표현형 1-3의 특징이다. 침략 챔버 광범위하게 암 세포 침윤 및 이동을 연구하기 위해 체외 도구로 사용되었고, 생체 내 종양 침윤과 전이의 메커니즘에 관한 지식을 제공 할 수있다. 챔버는 세포 배양 플레이트의 웰 내에 중첩 원통형 세포 배양 용 인서트로 구성된다. 인서트 하의 정의 기공 크기의 반 - 투과성 폴리 카보네이트 나 폴리스티렌 막이다. 표준 침공 분석에서, 세포는 혈청 무료 미디어를 삽입 막에 접종하고 혈청 또는 혈청과 같은 화학 유인 물질로 가득 세포 배양 우물에 배치 s의등 최대 chemoattractive 힘. 이 힘 드라이브의 세포를 검출하고 세포 수를 정량화하는 종래의 염료를 사용하여 염색 할 수있는 세포 외 기질 (침입)의 코팅을 통해 대안 반투막 (마이그레이션)을 통해 이동하거나한다. 세포 수는 다음 비 침습적 세포주의 이동과 침략의 정도에 따라 정규화된다. 이 방법은 연구자가 유전자 조작을 포함한 조건, 다양한에서 다른 세포 유형의 침략 가능성을 평가할 수 있습니다. 호르몬, 사이토 카인 및 성장 인자는 혈청의 중요한 구성 요소이다 점점 침습적 표현형 4 구동과 연관되는 것으로 도시되고있다. 그러나, 여전히 침략을 매개 이러한 chemoattractive 혈청 분자의 성격, 역할, 특이도는 애매 남아있다. 문제는 운전에 대한 책임이 있습니다 혈청 또는 혈청과 같은 화학 유인 물질의 특정 요인이 무엇인지 결정하기 위해 남아분자가 침입 과정을 억제 할 수 어떤 암세포 침윤뿐만 아니라. 이 보고서에서, 우리는 암세포의 침윤 및 chemoattraction 구동 분자로서, 호르몬, 사이토 카인 및 / 또는 혈청에 존재하는 성장 인자의 잠재적 개입을 평가하는 방법을 설명한다. 제안 된 프로토콜의 변형 된 목탄 스트립 핑은 2 % 목탄 스트립 FBS (CS-FBS)을 생성하기 위해 2 % 소 태아 혈청 (FBS)에서 호르몬, 사이토 카인 및 성장 인자를 제거하는 데 사용된다. 2 % FBS와 2 % CS-FBS 모두 암 세포 침공을 구동 chemoattractive 힘을 설정하는 에이전트로 사용됩니다. 통상 2 % FBS와 비교 chemoattractive 제로서 2 % CS-FBS를 사용하여 제 등 여러 가지 이점을 수득하고, 가장 두드러지게, 호르몬, 사이토 카인 및 성장 인자의 감소 / 상대 부재 하에서 암 침윤 표현형을 연구 할 수있는 능력. 또한, 호르몬, 성장 인자 집단 제거 여부를 평가하기 조사자 가능하며, CYtokines은 침략 지수의 증가 또는 감소가 발생합니다. 분석 후, (도 2 및 3 참조) &quot;X&quot;로 표시된 각각의 성분의 첨가는, 감소, 증가를 억제하거나, 원래 값으로 침습적 인덱스를 복원 할 수 있는지 여부를 결정하도록 설계된다. 이 방법론은 목탄 스트립 핑 동안 혈청으로부터 제거 성분의 생리 학적 수준을 달성하기위한 구체적인이지만, 또한 목탄 스트립 핑은 또한 신호 전달 경로에 영향을 미칠 수 있음을 유의해야한다. 예를 들면, 목탄 스트리핑 osteoprogenitor 세포의 알칼리성 포스파타제 활성을 감소 및 MAPK의 활성화 제 (5)의 환원을 통해 지방 형성을 유도 할 수 있다고보고되었다. 숯 박탈 미디어 시판하고 그 프로토콜에 따른 덱스 트란이 숯불을 겸비한 소 태아 혈청 O / N 항온 6 나와있다. 이 분석은 RESPO 개발되었다NSE는 주커 등. (7)에 의해보고 된 결과에 저자는 보통 2 % FBS와 미디어를 향해 마이그레이션 할 때 간극 결합 통신에 영향을 미치는 돌연변이 표현형 침공 지수의 증가를 보여 것을 보여 주었다. 이 증가는 숯불 제거 혈청의 교체와 함께 탈락했다. 이 결과에서, 저자들은 결론 지었다 친 유성 분자 향해 돌연변이의 영향이 이주 (구체적으로, 호르몬, 성장 인자, 사이토 카인 또는) 7. 이곳은 호르몬, 성장 인자, 사이토 카인 및 종양 촉진 및 내습 4에 관련된 신호 전달 경로 매개하는 것으로 알려져있다. 따라서, 과학 연구자가 어떤 요인 (들)이 연구에서 자신의 특정 암 세포 또는 돌연변이 종양 침략을 주도하고 결정하는 것이 중요하다. 성분의 농도 차이에 따라 결정되는 바와 같이 분석은 생리 학적 농도에서의 개별 성분의 역할을 충족하도록 설계 및# 8220; X &quot;정상 FBS와 CS-FBS에서. 이 분석의 개발을 통해, 연구자들은 자신의 시스템에 적용되는 특정 침입 경로에 더 많은 통찰력을 얻을 수 있습니다. 호르몬, 성장 인자, 사이토 카인은 종종 종양 촉진제 (8)으로 분류되어왔다. 이러한 EGF 이러한 요인 중 일부는 침략 챔버 9 화학 유인 물질에 대한 직접 소스로 사용된다. 따라서, 이러한 직접 종양 침윤이 혈청의 주요 구성 요소를 나타내는 것으로 보인다. 이 프로토콜은 조사자가 암세포의 잠재적 침입을 매개 호르몬, 성장 인자, 사이토 카인의 참여를 평가할 수 종래 내습 시험 관내 분석에 간단하면서도 중요한, 변경을 제안한다. 그러나, 분석은 귀중한 시간 및 R을 사용하지 않도록, 절차는 초기 분석 단계에서 그들의 연구에 효과적인 것인지에 대답 사관을 제공하도록 설계esources 불필요한 것으로 간주합니다. 이 방법은 CS-FBS를 사용하고 잠재적으로 암 세포 침공을 중재 리드 후보로 추구하는 분자있는 상태로 연구자의 판단에 의존한다. 이들 분석의 결과는 혈청 성분이 특정 세포주 또는 돌연변이에 대한 화학 유인 물질 또는 억제제가 연구되고있는 역할을 식별하는 데 유용하다는 것을 증명한다. 또한,이 방법은 조사 홍보 또는 암 세포 침윤을 억제하거나 키 신호 전달 경로를 식별하는 데 도움이 될 수 있습니다; 따라서 미래의 약물 디자인을 연출.

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

혈청 암세포 미세 혈관에 자신 intravasation 용이 마이그레이션 침입하는쪽으로 화학 유인로서 기능한다. 그러나, 세포가 이동하는 방향으로 실제 분자는 애매 남아있다. 이 변형 침윤 분석은 세포 이동 및 침입을 구동 대상을 식별하기 위해 개발되었다. 이 기술은 특정 호르몬, 성장 인자, 사이토 카인 또는 암세포의 잠재적 침입을 매개하는 역할을한다 여부를 확인하는 세 조건 하에서 내습 인덱스를 비교한다. 이러한 조건은 ⅰ) 정상 소 태아 혈청 (FBS)이, ⅱ) 숯 막았 호르몬, 성장 인자, 사이토 카인 및 ⅲ) CS-FBS + 분자를 제거 FBS (CS-FBS가), ( &quot;X&quot;)를 표시 포함한다. FBS 비교하여 CS-FBS와 세포 침윤에 큰 변화는 변화를 매개 호르몬, 사이토 카인 또는 성장 인자의 관여를 나타낸다. 개별 분자는 다시 자신의 능력을 분석하기 위해 CS-FBS에 다시 추가 될 수있다부르면 또는 침략 표현형을 구출. 또한, 두 개 이상의 요소가 구동 또는 첨가제 또는 침입을 억제하는 다수의 분자의 상승 효과를 평가하기 위해 결합 될 수있다. 전반적으로,이 방법은 호르몬, 사이토 카인, 및 / 또는 성장 인자가 암세포의 특정 형식 또는 특정 돌연변이에 대한 화학 유인 물질 또는 침입의 억제제로서 작용하여 세포의 침윤에서 역할을 수행할지 여부를 결정하는 조사원을 가능하게한다. 특정 화학 유인 물질 및 억제제를 확인함으로써,이 수정 침공 분석은 암 세포 침공을 지시 신호 전달 경로 규명하는 데 도움이 될 수 있습니다.

내습 인덱스는 비 침습적 세포주 정규화에 따라 각 조건에 대해 계산된다. 실험을 위해, 우리는 1205Lu 흑색 종 세포주 설립 변이체 안정한 세포주 우리 침습 선으로뿐만 아니라 1205Lu 셀들은 논리적 제어 역할 10 유도되었던 암성 비침 변이체 WM793를 사용한다. 즉 진피의 주성분이기 때문에 또한 내습 행렬로서 콜라겐 I를 이용한다. 최적 내습 행렬 세포주에 기초하여 생체와의 일치의 ...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

a-modified-vitro-invasion-assay-to-determine-potential-role-hormones

Research

JoVE Journal

Medicine

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

10.6K 조회수.  Roswell Park Cancer Institute. This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

비디오: A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

암 세포에 의해 내피 세포 단층의 침략을 측정하는 실시간 전기 임피던스 기반 기술

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

연구

의학

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

체외에서</em전이성 종양 성장에 휴면하고 스위치를 연구하기> 시스템

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

형성 초점 : 세포 기반 분석은 유전자의 발암 성 가능성을 파악하기 위해

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...