We are thankful to Dr. Richard Cerione, Dr. Marc Antonyak, and Kelly Sullivan, Cornell University, for providing technical advice, and to Dr. Gerlinde Van de Walle, Cornell University, for sharing their Olympus CKX41 inverted microscope.

3 % 아가 로스 2- 히드 록시 에틸의 제조 1. <ol><li> 깨끗하고 건조한 100 ml의 유리 병에, 30㎖의 증류수이어서 2- 히드 록시 아가 (아가 로스 VII) 0.9 g을 추가한다. </li><li> 15 초 부드럽게의 혼합물이 소용돌이 전자 레인지. 아가로 오스 분말이 완전히 용해 될 때까지이 단계를 적어도 세 번 이상 반복합니다. </li><li> 15 분 동안 용액을 함유 한 병을 압력솥. </li><li> 아가로 오스 솔루션을 추가로 사용하기 전에 실온까지 냉각 할 수 있습니다. 실온에서 솔루션을 저장합니다. </li></ol> 아래 층의 2. 준비 : 0.6 % 아가 로스 젤 <ol><li> 취급시 피펫에 응고에서 아가로 오스를 방지하기 위해 5 ㎖와 37 ° C 배양기에서 10 ml의 피펫 사전 따뜻한 몇 가지. </li><li> 부분적으로 병 뚜껑 마이크로파 15 초 동안 미리 만들어진 3 % 2- 히드 록시 아가로 오스 용액을 느슨하게. 그런 다음, 부드럽게 또 다른 15 초에 대한 솔루션과 전자 레인지를 소용돌이 친다. 주의 : 아가 소용돌이 때주의자체 솔루션 용액이 공기에 노출 이상 유출 할 수있을 때까지 상승하기 때문이다. </li><li> 병에 잔류 고체 젤, 몇 초 동안 전자 레인지가있는 경우. </li><li> 조기 고화 아가 용액을 방지하기 위해 다음 단계에서 45 ° C의 물 중탕에서 아가로 오스를 포함하는 용액을 보관 용기. </li><li> 37 ° C의 물을 욕조에 따뜻한 MCF10DCIS 미디어. 참고 : MCF10DCIS 미디어는 DMEM / F12, 5 % 말 혈청, 5 % 페니실린 스트렙토 마이신으로 구성되어 있습니다. </li><li> 전송 멸균 50 ㎖ 원뿔형 튜브에 예열 피펫을 사용하여 3 % 아가 로즈 용액 3 ㎖. </li><li> 즉시 따뜻한 MCF10DCIS 미디어의 12 ml에 추가하고 부드럽게 미디어와 아가로 오스를 혼합 원뿔 튜브를 반전. 그것은 식민지 나중에 계산을 방해하므로 모든 거품을 형성하지보십시오. </li><li> 부드럽게 기포를 형성하지 않고 6 웰 배양 플레이트의 각 웰에,이 혼합물을 2 ㎖를 추가한다. </li><li> 6 웰 배양 판 horizo​​nta을 품어에서야 1 시간 동안 4 ° C에서 평평한 표면에 혼합물이 응고 할 수 있도록합니다. </li><li> 혼합 응고 후, 30 분 동안 37 ° C를 인큐베이터에 접시를 놓습니다. 바닥 층을 사용할 준비가 된 것입니다. </li></ol> 셀 함유층 3. 제조 : 0.3 % 아가로 스겔 <ol><li> 를 Trypsinize MCF10DCIS 세포를 4 × 104 / ml의 세포 농도로 희석. </li><li> 멸균 50 ML 원뿔 튜브에 미리 예열 피펫 및 전송을 사용하여 3 % 아가로 오스의 2 mL를 취하여. </li><li> 즉시 원뿔 튜브에 MCF10DCIS 미디어의 8 ml에 추가하고 부드럽게 미디어와 아가로 오스를 혼합하는 반전. 모든 거품을 형성하지 마십시오. </li><li> (0 μM (DMSO) 또는 1 μM)을 MCF10DCIS 세포 (4 × 104 / ㎖) 2 mL를 취하여 BB-CLA로 처리. </li><li> 1 : 1 희석, 0.6 % 아가로 오스와 세포를 섞는다. </li><li> 세포 혼합물을 아가로 1 ㎖를 취하여 부드럽게 6 웰 배양 (P)의 바닥층 상에 추가후반 (2 × 104 세포 / ㎖). </li><li> 상단 층이 응고 할 수 있도록 적어도 15 분 동안 4 ° C에서 평평한 표면에 수평으로 6 웰 배양 판을 놓습니다. </li><li> 혼합 응고 후, 공급 층을 추가하기 전에 일주일 동안 37 ° C의 인큐베이터에 접시를 놓습니다. </li></ol> 세포층 4. 준비 : 0.3 % 아가 로스 젤 <ol><li> 전자 렌지 15 초 동안 미리 만들어진 3 % 2- 히드 록시 아가 용액. 부드럽게 다른 15 초에 대한 솔루션과 전자 레인지를 소용돌이 친다. </li><li> 45 ° C의 물을 욕조에 아가로 오스 솔루션 병 평형. </li><li> 37 ° C의 물을 욕조에 MCF10DCIS 미디어를 따뜻하게. </li><li> 50 ML 원뿔 튜브에 따뜻한 MCF10DCIS 미디어의 9 ml의 3 % 아가로 오스 액 1ml를 섞어 부드럽게 미디어와 아가로 오스를 혼합하는 반전. 공기 방울을 형성하지 마십시오. </li><li> BB-CLA와 혼합물 (0 μM (DMSO) 또는 1 μM)을 치료. </li><li> 부드럽게에 대한없이 (이 혼합물 1 ㎖를 추가밍 바닥 연질 층을 포함하는 6 웰 배양 플레이트의 각 웰에) 거품. </li><li> 혼합물이 응고 할 수 있도록 적어도 15 분 동안 4 ° C에서 평평한 표면에 수평으로 6 웰 배양 플레이트를 놓습니다. </li><li> 피더 층이 굳은 후, 37 ° C 배양기에 접시를 놓습니다. </li><li> 콜로니 형성이 관찰 될 때까지 새로운 매체를 사용하여 세포를 보충하기 위해 기존의 세포층에 0.3 % 아가로 오스 / 중 / 처리 용액 1 ㎖를 중첩하여이 공급 절차 주간를 반복합니다. 주 : 한천 부드럽고 피더 레이어 것은 따라서, 피더 레이어에서 추가 영양분 용이 세포에 도달하는 셀 - 함유 층으로 확산되며, 매우 부드럽고. </li></ol> 5. 데이터 수집 <ol><li> 소프트 한천에 세포 성장 2.5 주 후, 각 웰을 광학 현미경을 사용하여 콜로니 수를 카운트. 정량을 용이하게하기 위해, 투명도에 그리드를 인쇄에 그리드를 부착6 웰 플레이트는 세포가 계산하는 동안 어디에 찾을 수 있도록 도와줍니다. (각 식민지의 직경에 의해 정량화 등) 식민지의 크기가 달라질 수 있기 때문에, 채점되는 식민지 결정하는 기준 식민지 크기를 미리 정의. 예를 들어, 70 μm의 또는 데이터 분석의 큰 콜로니의 크기를 포함한다. </li><li> 더 콜로니 형성과 미래에 대한 계산을 방지하기 위해 4 ° C에서 샘플을 저장합니다. 이 건조 겔을 방지 파라 필름 6 웰 배양 접시를 밀봉. </li></ol>

<ol>
	<li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bioassay+of+human+tumor+stem+cells.">Primary bioassay of human tumor stem cells.</a> Science. 197, 461-463 (1977).</li><li>Wang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells.">Molecular signaling regulating anchorage-independent growth of cancer cells.</a> Mt Sinai J Med. 71 (6), 361-367 (2004).</li><li>Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., Pruijn, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD,+a+growing+family+of+citrullinating+enzymes:+genes,+features+and+involvement+in+disease.">PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.</a> Bioessays. 25 (11), 1106-1118 (2003).</li><li>Horibata, S., Coonrod, S. A., Cherrington, B. 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+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction.">Role for peptidylarginine deiminase enzymes in disease and female reproduction.</a> J Reprod Dev. 58 (3), 274-282 (2012).</li><li>Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R., Coonrod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis.">Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis.</a> Biochem Res Int. , 895343 (2012).</li><li>McElwee, J. L., 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=Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target.">Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.</a> BMC Cancer. 12, 500 (2012).</li><li>Knight, J. 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=Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney,+skin+and+vascular+disease+in+lupus-prone+MRL/lpr+mice.">Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.</a> Ann Rheum Dis. , 1-8 (2014).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> J Natl cancer Inst. 92, 1185-1186 (2000).</li><li>Fan, D., Morgan, L. R., Schneider, C., Blank, H., Fan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations.">Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.</a> J Cancer Res Clin Oncol. 109, 23-28 (2000).</li><li>Hamburger, A. W., White, C. P., Dunn, F. E., Citron, M. L., Hummel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum.">Modulation of human tumor colony growth in soft agar by serum.</a> Int J Cell Cloning. 1 (4), 216-229 (1983).</li><li>Anderson, S. N., Towne, D. L., Burns, D. J., Warrior, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+soft+agar+assay+for+identification+of+anticancer+compound.">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li></ol>

The authors have nothing to disclose.

소프트 한천 콜로니 형성 속도는 세포의 종류에 따라 달라 9. 따라서, 셀의 수는 최적화하고 그에 따라 조정되어야로 시작. 제안 된 초기 범위는 잘 6- 웰 플레이트를 이용하여 당 4~10 X 10월 2일부터 1일까지 X (5) 사이의 세포이다. 또한, 콜로니 크기는 각 셀의 성장 속도에 따라 변화한다. 따라서, 콜로니 크기 소정 컷오프 하류 정량적 분석에 대한 개별 콜로니 주석 필요하다...

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell&#8217;s ability to proliferate and form colonies in suspension within a semi-solid agarose gel1. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix2.
A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.
PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers6. For example, we have previously demonstrated that a &#8220;pan-PADI&#8221; inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids6. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies7. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein8. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

<table><tbody><tr><td>Zeiss Axiopot</td><td>Carl Zeiss Microscopy</td><td>1021859251</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza BioWhittaker</td><td>12-719F</td><td></td></tr><tr><td>HyClone Donor Equine Serum</td><td>Fisher Scientific</td><td>SH30074.03</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>2-Hydroxyethylagarose: Type VII, low gelling temperature</td><td>Sigma-Aldrich</td><td>39346-81-1</td><td></td></tr></tbody></table>

utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

소프트 한천 콜로니 형성 분석은 암세포의 종양 유발 문서화 광범위한 애플리케이션에 사용될 수있다. 이 기술의 주요 장점은 반고체 매트릭스 선택적 앵커리지 독립적 인 방식으로 증식 할 수있는 세포의 성장을 선호한다는 것이다. 이 특성은 주로 암세포가 아닌 일반 세포에 의해 발휘된다. 우리는 주로 약물에 의해 종양 성장 억제의 효과를 테스트하고 유방암 세포의 종양 유발에서 과발현 또...

Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

소프트 한천 콜로니 형성 분석의 활용은 유방암 세포에서 종양 유발의 억제를 확인하는

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

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28.4K 조회수.  Cornell University. Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. 

비디오: Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

NFκB 활동과 유방암 줄기 세포를 금지합니다 합성 및 아스피린 - 푸마 레이트의 전구 약물의 특성

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

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암 연구학

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.

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 ...

의학

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

종양 발생 세포 행동의 척도로 시험관 내 침략 분석에서 마우스 유방 종양 세포의 사용

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

유전학

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

낮은 중간 처리량에서 배양 포유류 세포에서 세포 성장 및 생존에 영향을 미치는 화합물을 식별하는 전략

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

생화학

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 ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

소프트 한천 오버레이 기법의 사용은 세균 생산 억제 화합물에 대한 화면으로

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

면역학 및 감염병학

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

소프트 한천 콜로니 형성 분석

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

생물학

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://www.jove.com/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

Soft Agar Colony Formation Assay: 암세포 증식에 대한 새로운 화합물의 효과를 테스트하는 방법

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

JoVE Encyclopedia of Experiments

Breast Cancer

In vitro studies

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Source: Kesarwani, M., et al. <a href="https://www.jove.com/t/58194">Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence.</a> J. Vis. Exp. (2019).
This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

메틸 셀룰로오스 기반 CFU 분석: 백혈병 세포의 집락 형성에 대한 항암 화합물의 효과를 확인하는 방법

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the ...