Name: labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies
Uploaded: 2016-11-30T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

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

<ol>
	<li>Jin, K., 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=Patient-derived+human+tumour+tissue+xenografts+in+immunodeficient+mice:+a+systematic+review.">Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review.</a> Clin Transl Oncol. 12 (7), 473-480 (2010).</li><li>Siolas, D., Hannon, 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=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Res. 73 (17), 5315-5319 (2013).</li><li>DeRose, Y. 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=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nat Med. 17 (11), 1514-1520 (2011).</li><li>Kabos, P., 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=Patient-derived+luminal+breast+cancer+xenografts+retain+hormone+receptor+heterogeneity+and+help+define+unique+estrogen-dependent+gene+signatures.">Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.</a> Breast cancer research and treatment. 135 (2), 415-432 (2012).</li><li>Zhang, X., 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=A+renewable+tissue+resource+of+phenotypically+stable,+biologically+and+ethnically+diverse,+patient-derived+human+breast+cancer+xenograft+models.">A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models.</a> Cancer Res. 73 (15), 4885-4897 (2013).</li><li>Lum, D. H., Matsen, C., Welm, A. L., Welm, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+human+primary+tumorgraft+models:+comparisons+with+traditional+oncology+preclinical+models+and+the+clinical+relevance+and+utility+of+primary+tumorgrafts+in+basic+and+translational+oncology+research.">Overview of human primary tumorgraft models: comparisons with traditional oncology preclinical models and the clinical relevance and utility of primary tumorgrafts in basic and translational oncology research.</a> Curr Protoc Pharmacol. , (2012).</li><li>Marangoni, E., 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=A+new+model+of+patient+tumor-derived+breast+cancer+xenografts+for+preclinical+assays.">A new model of patient tumor-derived breast cancer xenografts for preclinical assays.</a> Clin Cancer Res. 13 (13), 3989-3998 (2007).</li><li>Garrido-Laguna, I., 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=Tumor+engraftment+in+nude+mice+and+enrichment+in+stroma-+related+gene+pathways+predict+poor+survival+and+resistance+to+gemcitabine+in+patients+with+pancreatic+cancer.">Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer.</a> Clin Cancer Res. 17 (17), 5793-5800 (2011).</li><li>Landis, M. D., Lehmann, B. D., Pietenpol, J. A., Chang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+breast+tumor+xenografts+facilitating+personalized+cancer+therapy.">Patient-derived breast tumor xenografts facilitating personalized cancer therapy.</a> Breast Cancer Res. 15 (1), 201 (2013).</li><li>Norum, J. H., Andersen, K., Sorlie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+the+intrinsic+subtypes+of+breast+cancer+in+the+quest+for+precision+therapy.">Lessons learned from the intrinsic subtypes of breast cancer in the quest for precision therapy.</a> Br J Surg. 101 (8), 925-938 (2014).</li><li>Tentler, J. J., 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nat Rev Clin Oncol. 9 (6), 338-350 (2012).</li><li>Zhang, 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=Patient-derived+xenografts+of+triple-negative+breast+cancer+reproduce+molecular+features+of+patient+tumors+and+respond+to+mTOR+inhibition.">Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition.</a> Breast Cancer Res. 16 (2), R36 (2014).</li><li>Liu, 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cancer+stem+cell+metastasis+in+xenograft+animal+models.">Analysis of cancer stem cell metastasis in xenograft animal models.</a> Methods Mol Biol. 568, 7-19 (2009).</li><li>Thibaudeau, 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=Mimicking+breast+cancer-induced+bone+metastasis+in+vivo:+current+transplantation+models+and+advanced+humanized+strategies.">Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies.</a> Cancer Metastasis Rev. 33 (2-3), 721-735 (2014).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat Rev Cancer. 11 (2), 135-141 (2011).</li><li>Powell, E., 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=p53+deficiency+linked+to+B+cell+translocation+gene+2+(BTG2)+loss+enhances+metastatic+potential+by+promoting+tumor+growth+in+primary+and+metastatic+sites+in+patient-derived+xenograft+(PDX)+models+of+triple-negative+breast+cancer.">p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer.</a> Breast Cancer Res. 18 (1), (2016).</li><li>DeRose, Y. 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=Patient-derived+models+of+human+breast+cancer:+protocols+for+in+vitro+and+in+vivo+applications+in+tumor+biology+and+translational+medicine.">Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine.</a> Curr Protoc Pharmacol. , (2013).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. (32), (2009).</li><li>Indumathi, 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=Lineage+depletion+of+stromal+vascular+fractions+isolated+from+human+adipose+tissue:+a+novel+approach+towards+cell+enrichment+technology.">Lineage depletion of stromal vascular fractions isolated from human adipose tissue: a novel approach towards cell enrichment technology.</a> Cytotechnology. 66 (2), 219-228 (2014).</li><li>Hines, W. C., Yaswen, P., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer+requires+identification+and+correction+of+a+critical+cell+lineage-dependent+transduction+bias.">Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias.</a> Nat Commun. 6, 6927 (2015).</li><li>Campbell, J. P., Merkel, A. R., Masood-Campbell, S. K., Elefteriou, F., Sterling, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+bone+metastasis.">Models of bone metastasis.</a> J Vis Exp. (67), e4260 (2012).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+TGFbeta+Signaling+in+Mouse+Models+of+Cancer+Metastasis.">Imaging TGFbeta Signaling in Mouse Models of Cancer Metastasis.</a> Methods Mol Biol. 1344, 219-232 (2016).</li><li>Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J., Purchio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+human+breast+cancer+cell+lines+that+permit+rapid+and+sensitive+in+vivo+detection+of+mammary+tumors+and+multiple+metastases+in+immune+deficient+mice.">Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice.</a> Breast Cancer Res. 7 (4), R444-R454 (2005).</li><li>Lawson, D. A., 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=Single-cell+analysis+reveals+a+stem-cell+program+in+human+metastatic+breast+cancer+cells.">Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells.</a> Nature. 526 (7571), 131-135 (2015).</li></ol>

The authors have nothing to disclose.

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

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

<table><tbody><tr><td>DMEM/F12 (1:1)</td><td>Hyclone</td><td>SH30023.01</td><td></td></tr><tr><td>bFGF</td><td>BD Biosciences</td><td>354060</td><td></td></tr><tr><td>EGF</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H4784</td><td></td></tr><tr><td>B27</td><td>Gibco/Thermo Fisher</td><td>17504-44</td><td></td></tr><tr><td>Anti-fungi-antibiotics</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Accumax</td><td>Innovative Cell Technologies</td><td>AM-105-500</td><td>Digestion Buffer</td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>HBSS Red Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; free</td><td>Hyclone</td><td>SH30031.02</td><td></td></tr><tr><td>Hepes</td><td></td><td></td><td></td></tr><tr><td>10x PBS</td><td>Hyclone</td><td>SH30258.01</td><td></td></tr><tr><td>Cultrex</td><td>Cultrex</td><td>3433-005-01</td><td>Basement Matrix Extract (BME)</td></tr><tr><td>30 &amp;#176;C shaker</td><td>NewBrunswick Scientific CO. INC</td><td>Series 25 Incubator Shaker</td><td></td></tr><tr><td>70 &amp;#956;m filters</td><td>Falcon</td><td>7352350</td><td></td></tr><tr><td>scalpels</td><td>Fisher</td><td>22079690</td><td></td></tr><tr><td>Clorhexidine disinfectant</td><td>Durvet&amp;nbsp;</td><td>NDC# 30798-624-35</td><td></td></tr><tr><td>Red blood&amp;nbsp; cell lysis reagent</td><td>Sigma</td><td>R7757</td><td></td></tr><tr><td>Neuraminidase</td><td>Sigma</td><td>N7885-1UN</td><td></td></tr><tr><td>EpCAM (CD326+) microbeads*</td><td>Miltenyil Biotec</td><td>130-061-101</td><td></td></tr><tr><td>Lineage cell depletion Kit, mouse*</td><td>Miltenyil Biotec</td><td>130-090-858</td><td></td></tr><tr><td>MiniMACS Separator&amp;nbsp;</td><td>Miltenyil Biotec</td><td>130-042-102</td><td></td></tr><tr><td>Mini MACS Magnetic Stand</td><td>Miltenyil Biotec</td><td>130-042-303</td><td></td></tr><tr><td>MS Columns</td><td>Miltenyil Biotec</td><td>130-042-201</td><td>MS or LS columns can be used, adjust to number of cells.</td></tr><tr><td>Illumatool Tunable light system</td><td>Lightools research</td><td>Various</td><td>For in vivo fluorescence imaging</td></tr><tr><td>Xenogen IVIS200 imaging device</td><td>Xenogen</td><td>Various</td><td>For in vivo luminiscence imaging</td></tr><tr><td>Human Cytokeratin Clone MNF116 Monoclonal antibody</td><td>DAKO</td><td>M0821</td><td>Pan-cytokeratin&amp;nbsp;</td></tr><tr><td>Epidermal Growth factor receptor antibody</td><td>Cell signaling</td><td>4267S</td><td>EGFR</td></tr></tbody></table>

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

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

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

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

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

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

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

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

labeling-breast-cancer-patient-derived-xenografts-with-traceable

Research

JoVE Journal

Cancer Research

12.1K 조회수.  University of Colorado Anschutz Medical Campus.  우리는 녹색 형광 단백질과 루시 페라 제 기자를 발현하는 렌티 바이러스 입자 환자 유래의 이종 이식 (PDXs)의 안정적인 표시하는 방법을 설명합니다. 이 방법은 주 사이트 PDXs의 성장을 추적뿐만 아니라, 생체 내 이미징 시스템에서 사용 자발 실험 전이 검출을 허용한다. 

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

방사성 핵 종 형광 취재 원 유전자 설치류 종양 모델에서 종양의 진행을 추적 하기 위해 이미징

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

연구

암 연구학

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

암 생체 현지화위한 작은 동물의 계산된 Tomography 주 도형 시간 도메인 미만성 형광 Tomography

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

의학

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

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

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>orthotopically 주입된 유방 종양 세포의 자발적 폐 전이를 측정하기 위한 In vivo imaging

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

생체 내 Orthotopically injected breast tumor cells의 자발적 폐 전이를 측정하기 위한 이미징

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

폐 전이로부터의 종양 세포 전파 검사

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

저자 스포트라이트: 유방암 전이를 추적하기 위한 새로운 생체 내 기법을 사용하여 전이에서 전이로의 시딩 해독 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

<meta charset="utf8"></head><body>High-Throughput Dissociation and orthotopic Implantation of Breast Cancer Patient-derived Xenografts(유방암 환자 유래 이종이식편의 High-Throughput Dissociation and orthotopic Implantation of breast Cancer Patient-derived Xenografts)

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

<meta charset="utf8"></head><body>High-Throughput Dissociation and orthotopic Implantation of Breast Cancer Patient-derived Xenografts(유방암 환자 유래 이종이식편의 High-Throughput Dissociation and orthotopic I

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

High-Throughput Dissociation and orthotopic Implantation of Breast Cancer Patient-derived Xenografts(유방암 환자 유래 이종이식편의 High-Throughput Dissociation and orthotopic Implantation of breast Cancer Patient-derived Xenografts)

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

방사성 핵 종 형광 취재 원 유전자 설치류 종양 모델에서 종양의 진행을 추적 하기 위해 이미징

암 생체 현지화위한 작은 동물의 계산된 Tomography 주 도형 시간 도메인 미만성 형광 Tomography

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

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

생체 내 Orthotopically injected breast tumor cells의 자발적 폐 전이를 측정하기 위한 이미징

생체 내 Orthotopically injected breast tumor cells의 자발적 폐 전이를 측정하기 위한 이미징

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

광변환을 사용한 폐 전이에서 종양 세포 전파 추적

High-Throughput Dissociation and orthotopic Implantation of Breast Cancer Patient-derived Xenografts(유방암 환자 유래 이종이식편의 High-Throughput Dissociation and orthotopic Implantation of breast Cancer Patient-derived Xenografts)