이 작품은 미국 심장 협회 (American Heart Association) GRA 봄 09 박사후 (09POST2250268)와 NIH R01 CA097465에 의해 부분적으로 지원됩니다. 우리는 매우 종양 주입에 그녀의 도움 박사 웬 리 박사 VIKAS Gulani MRI 프로토콜 테스트 및 설정, 그리고 양 이본 파커를 주셔서 감사합니다.

프로토콜은 이전의 연구 11에서 적응. 1. CLT1 펩타이드에 GD-DOTA의 활용 <ol><li> 표준 고상 펩티드 합성을 사용하여, 2 - 클로로 트라이 틸 클로라이드 수지 (1.0 밀리몰)를 고체상 합성에 보호 된 아미노산으로부터 CLT1 펩티드 (CGLIIQKNEC)를 합성한다. </li><li> 최종 아미노산을 추가 한 후, 2 시간 동안 0 ℃에서 DMF의 탈륨 (III) 트리 플루오로 아세테이트 (1.09 g, 2.0 밀리몰, 2 당량)와 온 - 수지 선형 펩타이드 (20 ㎖)을 고리 화. </li><li> 다음에, 공액 PEG와의 Fmoc-NH-PEG-COOH (3.0 밀리몰)를 고체상 합성리스 (의 Fmoc)-OH (3.0 mmol)을 순차적으로 반응시켜 수지에 CLT1 펩티드 (1.0 밀리몰)의 N-말단에 라이신 순차적 및 고체상 합성리스 (의 Fmoc)-OH (6 밀리몰)의 두 번째 배치. </li><li> 피 페리 딘과 고체상 합성을 제거합니다. DOTA - 트리스 (T-샘프) (4.58 g, 각 아미노기 8.0 밀리몰, 2 당량)를 2 시간 동안 수지에 라이신 덴드리머에서 4 개의 무료 아민과 반응을 추가합니다. </li><li> 마지막으로, 절단 및 탈 보호 제품트리 플루오로 아세트산, 물, 그리고 실온에서 8 시간 동안 triisobutylsilane (TIS) (20 ㎖, 95/2.5/2.5)의 칵테일로 처리하여 수지에서. 트리 플루오로 아세트산 (TFA)로 세척 한 다음 여과하여 수지를 제거한다. 결합 된 여과 액이 차가운 에틸 에테르 (200 ㎖), 원심 분리기에 적가 추가, 에틸 에테르 배, 세척. 무색 고체 (1.99 g, 수율 61 %)을 수득 진공 하에서 말린다. </li><li> 10 분 동안 용매에 20 분 및 40-90% 용매 B위한 0-40% 용매 B의 구배 용매에서 (아세토 니트릴 중 0.1 % TFA) (0.1 % TFA 수용액)을 사용하여 분 취용 HPLC로 조 생성물을 정제 . </li><li> CLT1-DL-(DOTA) DI 물에 4 (250 ㎎, 0.077 mmol)을 (15 ml)에 용해시키고, 1 M 수산화 나트륨을 사용하여 pH를 6으로 조정합니다. 하나님 (OAC) 3 .4 H 2 O 추가 (472 밀리그램, 0.462 밀리몰, DOTA 모노 아미드 1.5 당량) 솔루션 부분에서, 1 M 수산화 나트륨을 사용하여 6시에 pH를 유지하면서. 48 시간 동안 RT에서 반응 용액을 교반 하였다. </li><li> 복잡한 전자와 잔류 하나님 (III)thylenediaminetetraacetic 아세트산 (EDTA) (90 ㎎, 0.31 밀리몰), 및 최종 생성물보기 CLT1-DL-(GD-DOTA) 4 (169 ㎎, 57 %)을 수득 크기 배제 컬럼으로 조질 생성물을 정제. </li></ol> 2. 대조 대리인의 특성 <ol><li> 유도 결합 플라즈마 광 방출 분광법 하나님 (III) 함량을 측정. </li><li> 매트릭스 2,5 - 디 히드 록시 산 (2,5-DHB)과 선형 모드에서 매트릭스 보조 레이저 탈착 / 이온화 비행 시간 (MALDI-TOF) 질량 스펙트럼을 획득. </li><li> relaxometer로 CLT1-DL-(GD-DOTA) 4의 조영제 및 nontargeted 조절제 60 메가 헤르츠 (1.5 T)에서 서로 다른 농도를 가진 sCLT1-DL-(GD-DOTA) (4)의 수용액의 휴식 시간을 측정 37 ° C에서 이 T를 측정하기 위해 500 에코와 T (1) 및 카 - 퍼셀-카르 · 퍼 어셀 · 메이 붐 · 길 서열을 측정하기 위하여 반전 복구 펄스 시퀀스를 사용한다. T 1과 T에게 일의 2 relaxivities을 계산하나님의 농도에 비해 1 / T 1과 1 / T 2의 플롯의 슬로프에서 전자 에이전트. </li></ol> 3. 동소 PC3 전립선 종양을 가진 동물 모델의 세포 배양 및 개발 <ol><li> / μL PBS 2.5 × 10 4 세포의 밀도 5 % 소 태아 혈청 및 페니실린 / 스트렙토 마이신 / fungizone, 트립신 처리하여 수확에 resuspend 보충 RPMI 배지에서 녹색 형광 단백질 (GFP)의 제정 식 문화 PC3 전립선 암 세포. </li><li> CWRU 기관 애니멀 케어 및 사용위원회에 의해 승인 된 동물의 프로토콜에 따라 무 흉선 동물 핵심 시설, 케이스 웨스턴 리저브 대학 (CWRU)에서 (4-5 주 이전) 남성 NIH 흉선 누드 마우스를 유지한다. </li><li> 수술 표면은 2 % 클로르헥시딘 용액을 분무 한 후 흡수성 커튼으로 덮여 있었다. 무균 수술 장갑이 절차를 수행하는 동안 착용했다. 동물 수술 전에 IP는 anesthetiz에 에버 틴 (250 ㎎ / ㎏)을 주입쥐를 전자. 대안 적으로, 케타민 / 자일 라진 / 아세 프로 마진은 50/5/1 ㎎ / kg의 농도로 사용될 수있다. 안과 용 연고 마취 중에 적절한 수분을 유지하는 마우스에 적용 하였다. </li><li> 멸균 메스를 사용하여 약 1 ㎝의 길이로 낮은 중간 선을 따라 피부와 복막을 통해 작은 절개를 절단하여 수술을 시작합니다. 무균 수술 드레이프가 생존 수술시 절개 주위에 덮여 있었다. </li><li> 조심스럽게 외면 화하다 전립선을 노출하는 전립선 등의 엽 (叶)을 안정화. 30 게이지 바늘을 전립선에 PBS (20 μL)에 PC3-GFP 세포의 현탁액을 주입한다. 마지막으로 12 autoclip 상처 절개를 닫습니다. </li><li> 시술 후 모니터링을 위해 1 주일 동안 하루에 두 번 동물을 모니터링 할 수 있습니다. 질병의 증상은 식사의 부족, 요도 주위에 어두운 소변 수집 및 스테이플에서 방광의 감염이나 폐색을 나타낼 수 불안정한 보행이 (가) 있습니다. </li><li> 10 일 이후, t 제거그는 스테이플 스테이플 리무버를 사용. 종양의 크기와 체중 감소, 행동 편차, 모터 결함으로 고통의 증거를 평가하기 위해 매일 동물을 모니터링합니다. 종양 성장 동안 통증 윤리적으로 허용 한계를 초과하거나 증거 종양 크기를 보였다 동물을 안락사. </li></ol> 4. 형광 이미징 및 조직학을 가진 펩타이드의 종양 결합 특이성 확인 <ol><li> 종양 세포 접종 후, 종양 성장의 약 사주 촬상로 시작하기 전에 허용한다. 종래 펩티드 탐침 주사로 종양의 존재를 확인하기 위해 형광 이미 저에서 방송 쥐 GFP 형광 이미지를 획득. </li><li> IV 텍사스 레드가 10 nmol의 / 마우스의 복용량에서 마우스 베어링 종양에 펩티드를 표시 주입. </li><li> 자궁 전위에 의해 나중에 마우스 2 시간을 희생, 종양 및 주요 장기를 수집하고, 형광 이미 저에 즉시 이미지. GFP (여기에 녹색 빛을 필터를 사용 : 444-490 뉴 멕시코, 방출 : 515 nm의 긴 패스 필터, 인수 설정 : 500-720 10 nm의 단계)와 텍사스 레드 (여기에 대한 붉은 빛 필터 : 576-621 nm의; 방출 : 635 nm의 긴 패스 필터; 획득 설정 : 630-800 10 nm의 단계). GFP와 텍사스 레드 150 밀리 초 10 밀리 초 노출 시간. </li><li> 즉시 전체 조직의 형광을 측정 한 후, 종양 조직을 수집 포르말린으로 고정하고, 동결 절단 (Cryosections) 5 μm의 조각으로. </li><li> PBS로 세정 한 후, 즉시 공 초점 레이저 스캐닝 현미경에 4를 함유하는 장착 매체의 한 방울 &#39;,6-diamidino-2-페닐 인돌 (DAPI), 화상 마운트. </li></ol> 5. 자기 공명 영상 (MRI) <ol><li> 약 사주 종양 세포 접종 후 종양이 직경 0.3 ~ 0.6 cm까지 자랄 수 있습니다. MRI 연구하기위한 음량 무선 주파수 (RF) 코일 (7) T MRI 스캐너를 사용한다. </li><li> 이소 플루 란 유도 챔버 내의 2 % 이소 플루 란 산소 혼합물로 마우스를 마취. OPHthalmic 연고 마취 중에 적절한 수분을 유지하는 마우스에 적용 하였다. </li><li> 헤파린 식염수로 채워진 1.0 m 길이의 튜브에 30 게이지 바늘을 삽입합니다. 마우스의 꼬리 정맥에 직접 만든 카테터를 놓습니다. </li><li> 자석에 마우스를 올려 놓고 코 콘을 통해 1.5 %의 이소 플루 란 산소와 흡입 마취 유지. </li><li> 속도와 호흡의 깊이를 모니터 할 수있는 복부의 모니터링 시스템에 연결된 호흡 센서를 놓습니다. 피드백 제어 시스템을 통하여 자석에 더운 공기를 불어 넣어 37 ° C에서 체온을 유지한다. </li><li> 종양의 위치 (TR / TE는 = 200/3.7 밀리 초, FOV를 = 식별하기 위해 지역화 시퀀스를 사용하여 시상 ​​부분 이미지로 시작하는 3.0 cm, 슬라이스 두께 = 2.2 mm, 슬라이스 번호 = 16, 평균 = 2, 플립 각도 = 45 °, 매트릭스 = 128 X 128). </li><li> CE-MRI (TR / TE = 151.2/1.9 밀리 초, FOV = 3.0 cm X 3.0 cm, 용 2D 축 이미지를 얻기위한 차원 T1 강조 그라데이션 지방 억제 시퀀스를 사용의이가 두께 = 1.2 mm, 슬라이스 번호 = 12, 평균 = 1, 플립 각도 = 80 °, 매트릭스 = 128 X 128). </li><li> 미리 주입 기준 MR 이미지 수집 한 후, 생리 식염수 200 μL로 세척하여 0.03 밀리몰 하나님 / kg의 투여 량에서 대상 에이전트 또는 제어 에이전트를 주입하기 시작한다. </li><li> 최대 30 분 동안 서로 다른 시간 지점에서 CE-MRI의 영상을 얻기 위해 계속합니다. </li></ol> 6. 이미지 처리 및 분석 <ol><li> 이미지 분석을 위해 이미징 소프트웨어를 사용합니다. </li><li> 두 차원 영상 평면에서 전체 종양에 관심 (로아)의 지역 및 신장을 그리고 평균 신호 강도를 측정한다. </li><li> (S 0 - ΔSNR = (S t / σ T) : CE-MRI 데이터를 정량화하기 위해, 다음과 같은 식을 이용하여 사전에 대비 SNR에 포스트 대비 SNR의 증가의 계산에 의한 종양 또는 신장 대비 향상 (ΔSNR)을 측정 / σ 0), S 0, S는 T 죽을 차례에 신호를 나타내는 곳또는 또는 신장 전과 대비, σ 0 및 σ의 t 후의 콘트라스트 전후 배경 공기로부터 측정 잡음의 표준 편차이다. </li><li> P &lt;0.05에서 통계적으로 유의 한 가정 학생의 양측 t-검정을 사용하여 P 값을 계산합니다. </li></ol>

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	<li>Brown, M. A., Semelka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MRI Basic Principles and Applications. , (2003).</li><li>Artemov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+magnetic+resonance+imaging+with+targeted+contrast+agents.">Molecular magnetic resonance imaging with targeted contrast agents.</a> J. Cell. Biochem. 90, 518-524 (2003).</li><li>Kalber, T. L., Kamaly, N., 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+low+molecular+weight+folate+receptor+targeted+contrast+agent+for+magnetic+resonance+tumor+imaging.">A low molecular weight folate receptor targeted contrast agent for magnetic resonance tumor imaging.</a> Mol. Imaging Biol. 13, 653-662 (2011).</li><li>Schmieder, A. H., Winter, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MR+imaging+of+melanoma+angiogenesis+with+alphanubeta3-targeted+paramagnetic+nanoparticles.">Molecular MR imaging of melanoma angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles.</a> Magn. Reson. Med. 53, 621-627 (2005).</li><li>Mulder, W. J., Strijkers, G. 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=MR+molecular+imaging+and+fluorescence+microscopy+for+identification+of+activated+tumor+endothelium+using+a+bimodal+lipidic+nanoparticle.">MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.</a> FASEB J. 19, 2008-2010 (2005).</li><li>Wang, S. J., Brechbiel, M., Wiener, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+new+MRI+contrast+agent+prepared+from+polypropyleneimine+dendrimers,+generation+2.">Characteristics of a new MRI contrast agent prepared from polypropyleneimine dendrimers, generation 2.</a> Invest. Radiol. 38, 662-668 (2003).</li><li>Amirbekian, V., Aguinaldo, J. G., 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=Atherosclerosis+and+matrix+metalloproteinases:+experimental+molecular+MR+imaging+in+vivo.">Atherosclerosis and matrix metalloproteinases: experimental molecular MR imaging in vivo.</a> Radiology. 251, 429-438 (2009).</li><li>Overoye-Chan, K., Koerner, 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=EP-2104R:+a+fibrin-specific+gadolinium-Based+MRI+contrast+agent+for+detection+of+thrombus.">EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus.</a> J. Am. Chem. Soc. 130, 6025-6039 (2008).</li><li>Pilch, J., Brown, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+selected+for+binding+to+clotted+plasma+accumulate+in+tumor+stroma+and+wounds.">Peptides selected for binding to clotted plasma accumulate in tumor stroma and wounds.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 2800-2804 (2006).</li><li>Rohrer, M., Bauer, H., Mintorovitch, J., Requardt, M., Weinmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+magnetic+properties+of+MRI+contrast+media+solutions+at+different+magnetic+field+strengths.">Comparison of magnetic properties of MRI contrast media solutions at different magnetic field strengths.</a> Invest. Radiol. 40, 715-724 (2005).</li><li>Wu, X., Burden-Gulley, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+peptide+targeted+small+molecular+Gd-DOTA+monoamide+conjugate+for+MR+molecular+imaging+of+prostate+cancer.">Synthesis and evaluation of a peptide targeted small molecular Gd-DOTA monoamide conjugate for MR molecular imaging of prostate cancer.</a> Bioconjugate Chem. 23, 1548-1556 (2012).</li><li>Burden-Gulley, S. M., Gates, T. 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=A+novel+molecular+diagnostic+of+glioblastomas:+detection+of+an+extracellular+fragment+of+protein+tyrosine+phosphatase+mu.">A novel molecular diagnostic of glioblastomas: detection of an extracellular fragment of protein tyrosine phosphatase mu.</a> Neoplasia. 12, 305-316 (2010).</li><li>McBride, J. 	. D., Birgit, H., Leatherbarrow, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resin-coupled+cyclic+peptides+as+proteinase+inhibitors.">Resin-coupled cyclic peptides as proteinase inhibitors.</a> Protein and Peptide. 3 (3), 193-198 (1996).</li><li>Cline, D. J., Thorpe, C., Schneider, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+method+for+facile+intramolecular+disulfide+formation+in+synthetic+peptides.">General method for facile intramolecular disulfide formation in synthetic peptides.</a> Anal. Biochem. 335 (1), 168-170 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

중요한 단계 적절한 바이오 마커 및 작은 펩티드를 표적의 선택 성공적으로 작은 크기의 대상 조영제를 개발하기 위해, 두 가지 중요한 점은 고려 될 필요가있다. 첫째, 정상 조직에 약간의 존재와 병에 걸린 조직에 풍부하게 존재하는 적절한 분자 생체를 선택하는 것이 중요합니다. 우리의 선택 암 관련 바이오 마커, 응고 된 혈장 단백질은 ?...

암 관련 분자 표적의 유효 촬상 이전 암 탐지와 진단의 정확성을 향상시키기 위해 큰 의미이다. 자기 공명 영상 (MRI)은 높은 공간 해상도와 더 이온화 방사선 1 강력한 임상 영상 기법이다. 그러나, 대상 조영제 임상 MR 암 분자 영상에 사용할 수 없습니다. 대상으로 MRI 조영제의 혁신적인 디자인과 개발은 크게 MR 암 분자 영상의 응용 프로그램을 진행한다. 상당한 노력이 암 세포의 표면에 발현 생체 MR 이미징을위한 대상으로 조영제를 개발하는되었습니다. 때문에 MRI 이러한 바이오 마커의 낮은 농도의 상대적으로 낮은 감도, 그것은 작은 분자 표적 조영제 2,3를 사용하여 효과적인 MR 분자 영상에 대한 충분한 대비 향상을 생성하는 도전이다. 충분한 향상을 얻기 위해서는, 다양한 전달 시스템 suc에성체의 하나님 (III) 킬레이트의 높은 페이로드 리포좀, 나노 입자와 고분자 복합체로 H 대상 사이트 4,5에서 조영제의 국소 농도를 증가시키기 위해 작성되었습니다. 이러한 전달 시스템은 동물 모델에서 중요한 종양 강화를 생성 할 수 있었지만, 그들의 큰 크기는 심각한 안전 문제 6의 원인이 독성 하나님 (III) 이온의 오랜 축적의 결과로, 몸에서 느리고 불완전한 제거로 만들었습니다. 최근 일부 연구는 분자 영상을위한 MRI의 한계가 병변에서 높은 지역의 표현으로 적절한 분자 바이오 마커를 선택하고 쉽게 7,8를 배출 할 수있는 작은 분자 에이전트를 사용하여 극복 할 수있는 것으로 나타났습니다. 이 에이전트의 주요 특징은 정상 조직에 약간의 존재와 병에 걸린 조직에 풍부하게 존재하는 분자 마커를 대상으로하고 있다는 것입니다. 조영제의 높은 농도는 충분한, 그 결과, 이러한 대상에 바인딩 할 수 있습니다효과적인 MR 분자 영상에 대한 cient 대비 향상. 그들의 크기가 신장 여과 임계 값보다 작기 때문에, 언 바운드 조영제 좀처럼 감소 배경 잡음으로 신체로부터 배출 될 수있다. 우리는 풍부하게 종양의 기질에 존재하는 보편적 인 암 관련 바이오 마커, 응고 혈장 단백질을 선택하고, 정상 조직 9 드물게 존재했다. 우리는 강한 PC3 전립선 암 모델 (10)에 특이 적으로 결합, 4 GD-DOTA 모노 아미드 킬레이트를 보였다 작은 표적 펩타이드 CGLIIQKNEC (CLT1)를 포함하는 대상 조영제를 합성. 여기, 우리는 생쥐의 종양을 감지하는 MR 암 분자 이미징을위한 방법론을 제공한다.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

종양 세포 외 기질 (extracellular matrix)은 암의 분자 이미징을위한 바이오 마커로 사용할 수 있습니다 암 관련 단백질의 풍부한 있습니다. 이 작품에서 우리는 종양의 기질에 응고 된 혈장 단백질에 특정 대상으로 MRI 조영제로 GD-DOTA 모노 아미드 복합 대상 작은 분자 펩타이드 효과적인 MR 암 분자 영상을 보여 주었다. 우리는 마우스 동소 PC-3 전립선 암 모델에서 MRI와 전립선 종양의 비 침습 검출 제의 효과를 평가하는 실험을 수행 하였다. 타겟 조영제 0.03 밀리몰 하나님 / kg의 낮은 투여 량으로 상당한 종양 콘트라스트 개선을 생산하는데 효과적이었다. MRI 조영제를 대상으로 펩타이드는 전립선 종양의 MR 분자 영상에 대한 약속한다.

도 1은 타겟 조영제 CLT1-DL-(GD-DOTA) 4 및 실험의 전반적인 구조의 합성을 도시한다. 임상 GD-DOTA (표 1)보다 CLT1-DL-(GD-DOTA) 4는 훨씬 더 완화도. 1.5 T에서 PBS에 CLT1-DL-(GD-DOTA) 4 (산도 7.4)의 가돌리늄 당 T 1 완화도는 GD-DOTA (10)보다 약 3 배 높은 수준이다. 비특이적이 (sCLT1-TR)는 (그림 2) 바인딩 아주 작은 종양을 보여줍니다 펩타이드 스크램블...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

쥐 전립선 암 모델에서 종양 침윤에 응고 된 혈장 단백질에 특정한 MRI 조영제를 대상으로 작은 펩티드와 암 MR 분자 이미징을 입증.

작은 분자 CLT1 펩타이드 표적 조영제와 전립선 암의 MR 분자 영상

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

mr-molecular-imaging-prostate-cancer-with-small-molecular-clt1

Research

JoVE Journal

Medicine

11.1K 조회수.  Case Western Reserve University.  쥐 전립선 암 모델에서 종양 침윤에 응고 된 혈장 단백질에 특정한 MRI 조영제를 대상으로 작은 펩티드와 암 MR 분자 이미징을 입증. 

비디오: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

염증성 플라크의 적외선 형광 (NIRF) Intravascular 분자 이미징, Multimodal 접근 가까운 생체내에서 죽상 경화증의 영상을

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

연구

의학

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

MicroCT를 사용하여 대비 향상된 베셀 영상

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

이식 근육 전구 세포를 타겟팅 할 수 분자 이미징

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

정화조 관절염의 경도 평가를위한 MR 표적 조영제를 사용하여 생체 내 대식 세포 이미징

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

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  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

동소 이식 췌장 암 마우스 모델의 동적 대비 강화 된 자기 공명 영상

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

전립선 암 환자의 발생은 종양 세포를 순환에서 이종 이식 모델을 파생

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

약물 치료를 종양 미세 환경을 공부하고 대상에 대한 뇌종양 / Organotypic 슬라이스 공동 문화 시스템

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...