이 작품은과과 (케빈 A. 로스와 SLC와 KRZ에 CA13148 - 35 SLC에 R01 CA122804, R01 CA134773) 신경계 질환과 뇌졸중의 국립 연구소 (SLC에 R01 NS048353), 국립 암 연구소에 의해 지원되었다 국방 (SLC에 X81XWH - 09 - 1-0086). UAB 종합 암 센터 스몰 애니멀 영상 공유 시설의 운영을 지원하는 자금은 NCI 코어 지원 교부금 (; E. 파트, PI P30 CA13148 - 35)에 의해 제공되었다. 우리는 알라바마 신경 과학 청사진 코어 센터 (P30 NS57098)과 도움을 UAB 신경 과학 코어 센터 (P30 NS47466)를 주셔서 감사합니다. 내용은 전적으로 저자의 책임이며 반드시 건강이나 국방부의 국립 연구소의 공식 견해를 대변하지 않습니다.

<ol>
	<li>Rosenberg, M. P., Bortner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+transgenic+and+knockout+animal+models+should+be+used+(for+drug+efficacy+studies+in+cancer).">Why transgenic and knockout animal models should be used (for drug efficacy studies in cancer).</a> Cancer Met. Rev. 17, 295-299 (1999).</li><li>Killion, J. J., Radinsky, R., 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=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Met. Rev. 17, 279-284 (1999).</li><li>Carroll, S. L., Ratner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+the+Schwann+cell+lineage+form+tumors+in+NF1?.">How does the Schwann cell lineage form tumors in NF1?.</a> Glia. 56, 1590-1605 (2008).</li><li>Woodruff, J. M., Kourea, H. P., Louis, D. N., Scheithauer, B. W., Kleihues, P., Cavenee, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology and Genetics of Tumours of the Nervous System. , 172-174 (2000).</li><li>DeClue, J. E. <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+receptor+expression+in+neurofibromatosis+type+1-related+tumors+and+NF1+animal+models.">Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.</a> J. Clin. Invest. 105, 1233-1241 (2000).</li><li>Byer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+inhibits+malignant+peripheral+nerve+sheath+tumor+growth+via+an+estrogen+receptor-independent+mechanism.">Tamoxifen inhibits malignant peripheral nerve sheath tumor growth via an estrogen receptor-independent mechanism.</a> Neuro-Oncology. , (2010).</li><li>Zinn, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+imaging+of+firefly+luciferase+reporter+gene+expression+in+skeletal+muscles+of+living+mice.">Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.</a> Mol. Therapy. 4, 297-306 (2001).</li><li>Luker, G. D., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+real-time+bioluminescence+imaging+for+the+study+of+viral+pathogenesis.">Luciferase real-time bioluminescence imaging for the study of viral pathogenesis.</a> Methods Mol. Biol. 292, 285-296 (2005).</li><li>Dahlberg, W. K., Little, J. B., Fletcher, J. A., Suit, H. D., Okunieff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitivity+in+vitro+of+human+soft+tissue+sarcoma+cell+lines+and+skin+fibroblasts+derived+from+the+same+patients.">Radiosensitivity in vitro of human soft tissue sarcoma cell lines and skin fibroblasts derived from the same patients.</a> Int. J. Radiat. Biol. 63, 191-198 (1993).</li><li>Badache, A., DeVries, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrosarcoma-derived+Schwann+cells+overexpress+platelet-derived+growth+factor+(PDGF)+receptors+and+are+induced+to+proliferate+by+PDGF.">Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF.</a> BB. J. Cell. Physiol. 177, 334-342 (1998).</li><li>Muir, D., Neubauer, D., Lim, I. T., Yachnis, A. T., Wallace, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+properties+of+neurofibromin-deficient+neurofibroma+Schwann+cells.">Tumorigenic properties of neurofibromin-deficient neurofibroma Schwann cells.</a> Am. J. Pathol. 158, 501-513 (2001).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 제시 자세한 orthotopic xenografting 방법은 우리가 널리 이러한 NF1 - 관련 주변 신경 칼집의 종양 연구에 사용되는 라인을 ST88 - 14 MPNST 전지를 사용하여 개발된 것이 하나이다. 그러나,이 방법은 다른 MPNST 셀 라인 잠복기 연구 쉽게 적응할 수 있습니다. 예를 들어, 우리는 우리가 ST88과 관찰과 비슷한 성공, STS - 26T 10 세포, NF1 - 관련 MPNST에서 파생됩니다 산발적으로 발생 MPNST 및 T265 - 2C 11 세포에서 파생된 라인 xenografting orthotopic 수행한 -14 세포. , 우리가 ST88 - 14 세포 xenografting orthotopic 여기 설명하는 정확한 조건이 직접 다른 MPNST 라인의 접목을 위해 채택 수 없습니다 갖는 것이라고 말했다. 대신, 방법론의 핵심 매개 변수는 경험적으로 각 세포주에 대한 결정되어야합니다. 이러한 매개 변수가 처음 이식할 MPNST 세포의 수, 낮은 정도 이식 개발과, 허용 시간을 포함, 볼륨이있는 종양 세포가 주입됩니다. 새로운 라인에 대한 이러한 매개 변수를 설정하기 위해, 우리 10월 3일부터 5일까지 X 10 6 종양 세포와 생쥐 (그룹 당 5 생쥐)의 그라 프트 그룹, 진도의 각 순서 (즉, 10 3 셀, 5 x는 세포의 두 농도를 확인 10 3 셀, 10 4 셀, 5 X 10 4 세포 등). 종양 세포 (&gt; 10 6)의 큰 숫자는 더 큰 볼륨에서 주입되어야하며 우리는 최대 5 ML에이 이식할 신경 세포의 손실없이 주입 수있는 것으로 나타났습니다. 우리는 또한 고밀도 suspensions이 종양 세포를 죽이는 전단하는 경향이 점점 증가함에 따라 5 ML 볼륨 당 더 이상 5 개의 X 10 6 종양 세포 주입 수있는 것으로 나타났습니다. 각 그룹 내에서 적어도 세 동물은 우리가 그 그룹을 종료하고 그래프트 성장을 확인하는 histologically 신경을 검토하는 시점에서 만져서 알 수있는 종양을 때까지 우리는이 생쥐를 따르십시오. 일반적으로, 우리는 30~60일 이내에 최대한 허용 종양의 성장에 도달할 것입니다 세포의 농도를 찾고 있습니다, 물론, 시간이이 시점은 주입된 세포의 수에 따라 달라집니다 도달할 필요합니다. 더 빠른 성장은 어려운 실험 및 / 종료하기 전에 효과적인 치료 농도를 달성하거나 실험 과정을 통해 충분한 bioluminescence 이미징 datapoints의 수집을 방지하기 위해 만들 수 있습니다. 시간 코스 이상 육십일보다 실험적인 매개 변수는 다루기 힘든 조정하게 그리고 불필요하게 계획된 실험 기간을 연장. 마지막으로, 우리는 우리가 tamoxifen 6 치료 효과를 입증하기 위해 NIH III 마우스로 이식할 ST88 - 14 세포이 orthotopic xenografting 방법론을 사용한 점에 유의합니다. 우리가 일상적으로 orthotopic xenografts에 후보 치료 대리인의 효과를 평가하는 데 사용하는 방법에 대한 자세한 설명은 그 논문에서 찾을 수 있습니다. 또한 neurofibroma 세포가 성공적으로 수정,이 프로토콜에 제시된 절차 neurofibromas에 대한 감독 후보 치료 요원들과 잠복기 실험을 수행하는 데 사용할 수있는, 그 제안, 말초 신경 12에 이식할 수있다는 증명되었습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Foxchase outbred SCID mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>#236</td>
<td>&nbsp;</td>
<tr>
<td>NIH III mice</td>
<td>&nbsp;</td>
<td>Taconic </td>
<td>#NIHBNX</td>
<td>&nbsp;</td>
<tr>
<td>NOD-SCID&gamma; mice</td>
<td>&nbsp;</td>
<td>Jackson Laboratories </td>
<td>#005557</td>
<td>&nbsp;</td>
<tr>
<td>Cell Stripper</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>25-056-cl</td>
<td>&nbsp;</td>
<tr>
<td>Vetbond surgical glue</td>
<td>&nbsp;</td>
<td>3M</td>
<td>1469SB</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton Company</td>
<td>#80030</td>
<td>10 &mu;L volume</td>
</tr>
<tr>
<td>Hamilton syringe needles</td>
<td>&nbsp;</td>
<td>Hamilton Company</td>
<td>#7803-05</td>
<td>33 Ga., 0.5 inch, PT4 (custom made)</td>
</tr>
<tr>
<td>Ear tags</td>
<td>&nbsp;</td>
<td>National Band and Ear Tag Co.</td>
<td>1005-1</td>
<td>&nbsp;</td>
<tr>
<td>Ophthalmic ointment</td>
<td>&nbsp;</td>
<td>Dechra</td>
<td>NDC 17033-211-38</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

체외 화면에서 후보 치료 요원의 초기 식별, 종양 성장이 적합한 동물 모델에서 수행되어야합니다 억제하는 약물의 능력의 엄격한 심사를위한 필수 있지만. 이러한 목적에 가장 적합합니다 동물 모델의 유형은 강렬한 토론의 주제입니다. 어떤 증거는 유전자 변형 생쥐에서 발생하는 종양에 대한 약물 효과를 검토 잠복기 재판은 임상 결과 1 개 중 더 예측하고 있으므로 치료 후보 대리인이 자주 이러한 모델 테스트는 것을 나타냅니다. 불행히도, 유전자 변형 모델은 다양한 종양 유형에 사용할 수 없습니다. 또한, 유전자 변형 모델은 종종 등은 인간의 대응, 종양 표현형 및 무능력의 불완전 penetrance은 종양이 개발 시기를 예측하는 방법을 잘 마우스 종양 모델에 같은 문제와 같은 다른 제한이 있습니다. 적절한 형질 종양 모델을 사용할 수없는 경우에는 그 결과, 많은 조사는 잠복기 실험에 대한 이종 이식 모델을 (인간의 종양 세포가 immunodeficient 생쥐에 이식할)을 사용합니다. 유전자 변형 모델을 사용할 경우에도 후자는 치료 범위의 빠른 결정을 촉진로서, 그들은 종종 이종 이식 모델와 제휴하고 있습니다. 또한,이 제휴는 유전자 변형 종양과 진정한 인간의 종양 세포에있는 에이전트의 효율성을 비교하실 수 있습니다. 역사적으로, 자주 (이소성 xenografts) subcutaneously 종양 세포를 주사에 의해 수행되었습니다 xenografting. 이 기술은 신속하고 상대적으로 저렴한, 재현할 수 있으며, 치료 기간이 중 종양 성장을 지속 quantitation 수 있습니다. 그러나, 피하 공간 이소성 xenografting로 얻은 가장 neoplasms 그리고 결과에 대한 일반 microenvironment하지 않습니다 인해 같은 호스트 조직 및 종양 유전자의 기관이 특정 표현의 부재로 요인에 오해하실 수 있습니다. 그것은 따라서 강력하게 이소성 이식 연구가 인간의 종양 세포가 원래 조직 (orthotopic xenografting) 2 융합하는 연구로 대체하거나 보완하는 것이 좋습니다되었습니다. 불행히도,이 권고의 구현은 종종 orthotopic xenografting 방법론이 아직 많은 종양 유형에 대한 개발되지 않은 사실에 의해 저지됩니다. 악성 말초 신경 쉬스 종양 (MPNSTs)는 neurofibromatosis 타입 1 3과 가장 일반적으로 좌골 신경 4 발생과 산발적으로 또는 조합에서 발생하는 매우 공격적인 sarcomas 있습니다. 여기 반딧불 루시페라제 - 태그 MPNST 인간 세포가 orthopically immunodeficient 생쥐의 좌골 신경에 xenografted되는 기술 직접적인 방법을 설명합니다. 개별 동물과 스터디 그룹에 후속이 아닌 편견 임의화에 접목 절차의 성공을 평가하는 우리의 방식도 설명되어 있습니다.

Watch this Scientific Journal Video about Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents at JoVE.c

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

안정 immunodeficient 생쥐의 좌골 신경에 루시페라제 - 태그 인간의 악성 말초 신경 칼집 종양 세포를 접목하는 방법이 설명되어 있습니다. 스터디 그룹에 동물 임의의 분리를위한 종양 이식과 기준의 적절한 설립을 증명하기 위해 bioluminescence 이미징의 사용도 설명합니다.

대한 인간 루시페라제 태그가 추가된 악성 주변 신경 쉬스 종양 세포 Orthotopic Xenografting 생체내에</em후보 치료 대리점> 테스트

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

orthotopic-xenografting-human-luciferase-tagged-malignant-peripheral

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Medicine

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

18.5K 조회수.  University of Alabama at Birmingham - UAB. 안정 immunodeficient 생쥐의 좌골 신경에 루시페라제 - 태그 인간의 악성 말초 신경 칼집 종양 세포를 접목하는 방법이 설명되어 있습니다. 스터디 그룹에 동물 임의의 분리를위한 종양 이식과 기준의 적절한 설립을 증명하기 위해 bioluminescence 이미징의 사용도 설명합니다.

비디오: Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

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 ...

연구

의학

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

마우스에 Intracerebroventricular을 통해 치료 대리점 납품 (ICV)과 정맥 (IV) 사출

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

종양의 4D 라이브 세포 이미징을위한 MAME 모델 : Microenvironment 상호 작용 영향을 미치는 악성 진행

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

인간의 전립선 암 전이의 동소 쥐과 모델

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomic 프로파일 의약품 응답 테스트를위한 3D 인간의를 Biogel 문화 시스템을 사용 Microtumors 및 환자 유래 아교 모세포종 세포의 생성

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

작은 동물에 대한 치료제의 폐 통기를위한 일회용 Dosators

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

대한 인간 루시페라제 태그가 추가된 악성 주변 신경 쉬스 종양 세포 Orthotopic Xenografting 생체내에 테스트