Research in Zhang lab was supported by X. H.-F. Z.'s NCI CA151293, CA183878, Breast Cancer Research Foundation, U.S. Department of Defense DAMD W81XWH-13-1-0195, a Pilot Award of CA149196-04, McNair Medical Institute and by H.W.'s U.S. Department of Defense DAMD W81XWH-13-1-0296.

모든 동물의 작업은 의학의 베일러 대학의 동물 관리 지침에 따라 이루어졌다. 1. 셀 준비 주의 : 다른 암 세포주는 IIA 주입은 연구 목적에 따라 사용할 수있다. 우리는 유방암 세포주를 사용한 MCF7, 4T1, 4T07, MDA-MB-361, MDA-MB-231, MDA-MB-436 및 연구에서 전립선 암 세포주 C4-2. 우리는 일반적으로 우리의 연구를 위해 GFP- 및 반딧불이 루시퍼 라제 표지 암 세포를 모두 사용하여 GFP - 루시 페라 표지 MCF7 세포 라인에서 여기에 몇 가지 데이터가 표시됩니다. <ol><li> 5 % CO 2에서 37 ℃에서 10 % FBS 및 0.1 ㎎ / ㎖의 페니실린 - 스트렙토 마이신을 함유하는 DMEM에서 세포를 유지한다. </li><li> 0.25 % 트립신 / EDTA 용액으로 90 % 합류 - IIA 주입하기 전에 80에서 세포를 Trypsinize. 셀 카운팅 10 ml의 PBS에서 셀을 두 번 세포를 씻어 다시 중단 차가운 PBS를 사용합니다. 5 분 동안 800 XG에서 PBS, 원심 분리기 세포를 제거합니다. </li><li> 세포를 다시 일시 중지빙냉 PBS에서 5 × 106 세포 / ml의 농도. </li><li> 100 μL의 GFP- 한 마우스의 IIA 주입을위한 반딧불이 루시퍼 라제 표지 암 세포를 사용합니다. 주 :. 따라서, 각각의 동물에 의해 수신 된 셀의 수는 5 × 5이 숫자는 세포주의 공격성에 기초하여 변경 될 필요가있다. </li><li> 얼음에 세포 현탁액을 유지합니다. 주 : 진동은 사출 할 준비가 될 때까지 몇 분마다 세포 응집을 방지하기 위해 필요할 수있다. (예 : MDA-MB-231와 같은) 일부 세포주 (예를 들어, 45 μm의 세포 여과기를 통과하는) 더 엄격한 절차는 응집을 방지하기 위해 필요할 수있다. 혈관의 막힘은 조직 괴사가 발생할 수 있습니다, 따라서 실험을 교란하시기 바랍니다. </li></ol> 2. 동물 준비 <ol><li> 동물의 통증 관리를 위해, 아니 이하 24 이상의 시간 수술 전에 쥐에 건강 보조 식품 5 ㎎ / ㎏ / 일 카프로 펜 (또는 다른 진통제)을 얻었다. 관리수술 전에 0.1 ㎎ / ㎖ 프레 놀핀 피하에 60 분의 용량. 참고 : 옵션 절차는 별도의 에스트라 디올을 제공하기 위해 동물의 뒷면에 에스트라 디올 팔레트를 이식하는 것입니다. 이는 ER + 암 세포 성장 촉진 (예 : MCF-7 세포) (9)을 가속한다. </li><li> 복강 내 주사하여 - (100 ㎎ / ㎏ 80)과 자일 라진 (10 -12.5 ㎎ / ㎖) 케타민 6 주 이전 마우스 (약 20g) - 4를 마취. </li><li> 발가락 핀치에 의해 20g 마우스와 호흡 속도 50 % 감소의 관찰의 진정 작용의 적절한 수준을 확인하고 점막 핑크 (예를 들어, 마우스 귀 피부 및 발 피부). 수술 중에 이러한 생체 신호 매 15 분 모니터링 유지합니다. 참고 : 동물의 어떤 움직임은 동물이 수술 충분히 마취와 준비가되어 있음을 나타냅니다. </li><li> 건조 함을 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> , 하복부에 특히 오른쪽 사타구니 영역을 마우스를 면도 곳 수술과 주사는 것발생한다. 참고 : 무 흉선 누드 마우스를 사용하는 경우이 필요하지 않습니다. </li><li> 그 뒷면에 마우스를 올려 그들의 자연적인 위치에 다리를 확산하고 테이프로 이동 해부 판지에 발가락 스틱. </li><li> 70 % 이소 프로필 에탄올을 적신 멸균 면봉와 오른쪽 사타구니 부위를 닦아, betadine 수술 스크럽 여러 번 하였다. </li></ol> 3. 일반 장골 정맥과 동맥의 위치 및 분리 <ol><li> 긴 4 번째와 3 번 핸들에서 개최 살균, # 10 탄소 강철 메스 블레이드를 사용하여 오른쪽 아래 복부에 5 일 젖꼭지 사이에 1.2 cm - 1.0의 피부 절개를합니다. 그 후 절개 부위를 제외하고는 동물의 시체를 충당하기 위해 무균 수술 드레이프를 사용합니다. </li><li> 표준 벤치 탑 해부 현미경으로 동물을 이동합니다. 주입을위한 4 배의 배율을 사용합니다. </li><li> 해부 현미경의 4 배 배율에서 지방 조직과 peritoneu 사이에 무딘 분리 집게를 삽입m, 그리고 장골 혈관과 신경 (그림 1 참조) 노출 양쪽에서 바깥쪽으로 조직을 밀어 넣습니다. 참고 : 중간 선에서 대동맥 및 동맥 가지의 아래 부분 사이의 동맥이 총장 골 동맥이라고합니다. 주입이 일어나는 곳이다. </li><li> 가까운 선박, 선박과 신경 사이 (막 등) 결합 조직을 돌파 똑바로 잘 포셉 한 쌍을 사용합니다. </li><li> 왼쪽에 직선 미세 집게를 전환합니다. 오른손을 사용하여 각도 미세 집게 한 쌍을 잡아. 결합 조직이 파괴되어 동일한 위치로 오른쪽 집게를 삽입합니다. 그리고 용기 아래 통해 오른손 집게 스틱. </li><li> 동시에 통과하는 오른손 집게를 안내하기 위해 용기의 왼쪽에있는 결합 조직에 왼손 집게를 삽입한다. </li><li> 오른손 집게 용기 아래 얻으면, 주변 TI로부터 용기를 분리하려고조금 더 ssue 한 다음 오른쪽 집게의 상단에 보관하십시오. </li><li> 오른쪽 집게에 4-0 실크 봉합사의 끝을 제공하기 위해 왼쪽 집게를 사용하고 용기 아래를 부드럽게 천천히 봉합사를 잡아 당깁니다. 참고 : 이제 모두 일반적인 정맥 및 동맥 혈관을 봉합에 의해 해제된다 (그림 2 참조). </li></ol> 4. 사출 및 후 분사 케어 <ol><li> 31 G 인슐린 주사기에 주입을위한 세포를 준비합니다. 주입 당 PBS에 세포 현탁액 100 ㎕를 사용합니다. 아래로 주입 된 세포를 막고 혈액 흐름을 차단하지 않도록 세포를 분리하고 응집을 방지하기 수회 피펫하고 확인. </li><li> 왼쪽 손에 각진 집게로, 집게에 부드럽게 개최 혈관을 갖는 봉합사의 안내에 따라 선박 아래에있는 집게를 넣어, 다음 집게의 두 팔을 엽니 다. 참고 : 집게의 두 팔 사이의 간격이 난 것입니다njection 사이트입니다. </li><li> 주입을위한 준비, 위쪽으로 바늘의 경사와 함께 오른쪽에 100 ㎕의 세포 현탁액로 31 G 바늘을 잡으십시오. </li><li> 동맥 루멘에 바늘을 삽입 (피가 바늘 끝을 입력합니다). 그런 다음 세포를 주입하는 천천히 세포 현탁액을 누릅니다. 세포 현탁액을 용기 내의 적혈구를 밀어 것이다. 혈관을 파괴하거나 세포 현탁액을 누설하지 않도록하십시오. </li><li> 주입이 완료되면, 조심스럽게 바늘을 철수하고 출혈을 멈추게하기 위해 왼쪽 집게에 선박을 들고 유지. 그런 다음 봉합을 당깁니다. </li><li> 왼쪽 집게을 당깁니다, 신속하게 출혈을 멈추게하기 위해 동맥 절개 영역을 누릅니다 면봉을 사용합니다. 참고 : 일반적으로 5-10 분 연속 압력이 출혈을 멈추게하기에 충분하다. </li><li> 중지 출혈하면 수술 영역의 피부 가장자리를 폐쇄 피부 접착제를 사용한다. 귀 태그를 확인하고 주입 된 세포의 분포를 조사하는 신호 생물 발광을 확인합니다. &lt;BR /&gt; 참고 : 성공적인 주입은 3 그림과 유사한 이미지가 발생합니다. <ol><li> 75 밀리그램 / 인트라 궤도 동을 통해 kg 마우스 농도로 PBS에서 / ㎖ 루시페린 100 ㎕를 15 mg을 주입하여 생물 발광 신호를 확인합니다. <ol><li> 내부 안와 주입 들어, 마우스 눈의 양쪽에 손가락을 넣어 안구 약간 눈 프레임으로부터 튀어 안구 코 사이 28 G 인슐린 주사기 바늘을 넣고 천천히 루시페린를 주입 주사기를 추진하기 위해 압력을 . 참고 : 올바른 위치에서, 바늘 (2)의 깊이에 도달 할 수 있어야합니다 - 경조직 (뼈)을 치기 전에 3mm를. </li><li> 10 생체 내 동물 전체 이미징 이미징 시스템에 마우스 플레이스 - 5 분 내에 60 초 (도 3 참조). 주 : 선택 사양 인 절차를 추가 에스트라 디올을 제공하기 위해 동물의 뒷면에 에스트라 디올 펠릿을 이식하는 것입니다. 이는 ER + 암 세포의 성장 (예를 들어, MCF-7 가속화셀) 15. </li></ol></li></ol></li><li> 이 깨어 때까지 따뜻한 가열 패드에 마우스를 둡니다. 포스트 수술 기간 동안 열개과 고통의 흔적을, 출혈을 모니터링 붓기. 고통의 흔적까지 통증 관리를 위해 주어진 진통 범위 (carprofin 건강 보조 식품 제안)와 케이지에 다시 마우스를 넣습니다. 수술 후 칠일 동안 동물에 세심한 관심과주의를 기울이십시오. </li></ol> 5. 모니터링 전이성 성장 <ol><li> 조직 형태 측정 : <ol><li> 뼈 조직을 베어링 수확 종양 후, 24 시간 동안 4 % 파라 포름 알데히드의 뼈를 고정 pH를 3 7.0 14 % EDTA 용액을 석회 - 오일을, 그리고 이전에 포함 (14)을 설명 파라핀 그들을 대상으로 할 수 없다. </li><li> 제 파라핀 포함 골이 3 μm의 두께 조직 및 이전 (14) 설명에 따라 헤 마톡 실린 / 에오신 염색의 표준 조직 학적 방법을 수행합니다. </li></ol></li><li> Immunohistochemis시험: <ol><li> 주제 파라핀 포함 된 종양 베어링 뼈 이전 14 설명 된 바와 같이 다양한 용도로 염색 면역 조직 화학 염색하는 슬라이드. 요약하면, 각각 항 Osterix 항체 및 항 알칼리 포스 파타 아제 (ALP) 항체에 의해 방지 GFP 항체, 및 발현 사이 골 형성 세포 (사전 조골 세포와 조골 세포)에 의해 MCF7 암 세포 발현 사이. </li></ol></li></ol>

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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=Tumor+microenvironment+regulates+metastasis+and+metastasis+genes+of+mouse+MMTV-PymT+mammary+cancer+cells+in+vivo.">Tumor microenvironment regulates metastasis and metastasis genes of mouse MMTV-PymT mammary cancer cells in vivo.</a> Vet Pathol. 51 (4), 868-881 (2014).</li><li>Xiang, 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=CXCR4+Protein+Epitope+Mimetic+Antagonist,+POL5551,+Disrupts+Metastasis+and+Enhances+Chemotherapy+Effect+in+Triple+Negative+Breast+Cancer.">CXCR4 Protein Epitope Mimetic Antagonist, POL5551, Disrupts Metastasis and Enhances Chemotherapy Effect in Triple Negative Breast Cancer.</a> Mol Cancer Ther. 14 (11), 2473-2485 (2015).</li><li>Wang, 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=The+osteogenic+niche+promotes+early-stage+bone+colonization+of+disseminated+breast+cancer+cells.">The osteogenic niche promotes early-stage bone colonization of disseminated breast cancer cells.</a> Cancer Cell. 27 (2), 193-210 (2015).</li><li>Hoffmann, 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=Characterization+of+new+estrogen+receptor+destabilizing+compounds:+effects+on+estrogen-sensitive+and+tamoxifen-resistant+breast+cancer.">Characterization of new estrogen receptor destabilizing compounds: effects on estrogen-sensitive and tamoxifen-resistant breast cancer.</a> J Natl Cancer Inst. 96 (3), 210-218 (2004).</li><li>Tannehill-Gregg, S. H., Levine, A. L., Nadella, M. V., Iguchi, H., Rosol, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+zoledronic+acid+and+osteoprotegerin+on+growth+of+human+lung+cancer+in+the+tibias+of+nude+mice.">The effect of zoledronic acid and osteoprotegerin on growth of human lung cancer in the tibias of nude mice.</a> Clin Exp Metastasis. 23 (1), 19-31 (2006).</li><li>Slyfield, C. R., Tkachenko, E. V., Wilson, D. L., Hernandez, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Dynamic+Bone+Histomorphometry.">Three-Dimensional Dynamic Bone Histomorphometry.</a> J Bone Miner Res. 27 (2), 486-495 (2012).</li><li>Koba, W., Jelicks, L. A., Fine, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroPET/SPECT/CT+imaging+of+small+animal+models+of+disease.">MicroPET/SPECT/CT imaging of small animal models of disease.</a> Am J Pathol. 182 (2), 319-324 (2013).</li><li>Simmons, J. 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=Canine+prostate+cancer+cell+line+(Probasco)+produces+osteoblastic+metastases+in+vivo.">Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo.</a> Prostate. 74 (13), 1251-1265 (2014).</li><li>Amend, S. R., 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=Thrombospondin-1+regulates+bone+homeostasis+through+effects+on+bone+matrix+integrity+and+nitric+oxide+signaling+in+osteoclasts.">Thrombospondin-1 regulates bone homeostasis through effects on bone matrix integrity and nitric oxide signaling in osteoclasts.</a> J Bone Miner Res. 30 (1), 106-115 (2015).</li></ol>

The authors have nothing to disclose.

만 장골 동맥 암세포 주입 대상이지만, 우리는 주변 조직 모두에서 장골 동맥과 정맥을 분리하는 것을 권장하고 번들로 이들을 함께 리프트. 정맥 및 동맥 광범위하게 서로 접촉하기 때문이고, 정맥 혈관 벽이 얇고 파괴하기 쉽다. 암 세포에서만 동맥에 주입되지만 따라서 성공적인 주사의 경우, 시간과 함께 두 개의 용기를 유지하는 노력을 절약. 도 2에 도시 된 바와 같이 4-0 실크 봉...

전이는 고형 종양에 의한 사망의 90 %를 차지한다. 뼈는 다양한 종류의 암, 특히 유방암과 전립선 암의 전이에 의해 영향을 가장 많이 기관이다. 병원에서 진단하면, 뼈 전이는 일반적으로 이미 자주 신경 학적 증상을 동반 뼈에 골 용해성 또는 골아 중 하나를 변경, 첨단 단계를 입력했습니다. 이전의 연구는 주로 그러나 우리는 현재 골 용해성 프로세스의 개시 전에 뼈의 미세 전이의 이해를 제한, 명백한 골 용해성 골 전이 1-3에 집중했다. 이는 적절한 실험 모델 및 방법 부족 적어도 부분적이다. 유방암 유전자 변형 마우스 모델들은 폐 전이 있지만, 훨씬 덜 효율적 뼈 4. 마찬가지로, 동소 이식 된 종양은 거의 어떤 뼈 열대 4T1 유방 carcinom으로, 자발적인 뼈 전이를 개발하지서브 클론 및 MSP는 예외 5-7로 PyMT 형질 전환 마우스 모델을 과발현. 인트라 경골 뼈 드릴 8-10에 암세포를 제공 할뿐만 아니라 지방 조직의 손상과 염증을 초래한다. 현재 유방암 세포주 내 심장 주입 뼈 정착 11-13 조사 주요 접근하고있다. 암세포 좌심실에 도입 된 후에 그러나 제한된 비율은 최종적 어려운 정량 방식으로 미세 전이를 추적 할 수있게 뼈 골수에 도달한다. 본 연구에서 우리는 선택적하여 지방 조직에 손상을주지 않고 뼈 골수 암 세포 농축, 뒷다리 조직에 암세포를 제공하도록하는 기술, 즉, 인트라 - 장골 동맥 (IIA) 주입 (14)을 설치한다. 때문에 뼈 특이성이 방법은 결국은 An 정착하기 전에 무통 암세포에 충분한 시간을 허용imals 다른 중요한 장기에 차 종양이나 전이에 굴복. 이러한 생물 발광 이미징, 면역 형광 염색 및 골 조직 형태와 같은 다른 기술의 다양한 결합하면 IIA 주입 특히 다중 셀에 하나의 암 세포의 진행을 추적하기 위해, 뼈 전이에 관한 연구 목적의 넓은 범위에 대한 잠재적으로 유용한 미세 전이. 특히, IIA 분사 암 세포 및 골 미세 셀 주변의 여러 유형의 상호 작용을 시각적으로 우리 수 있는지 보여 주었다.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>HyClone</td><td>SH30022.01</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Pen/Strep Amphatericin B</td><td>Lonza Biowhittaker</td><td>17-745E</td><td></td></tr><tr><td>PBS</td><td>Lonza Biowhittaker</td><td>17-516F</td><td></td></tr><tr><td>Trypsin/EDTA solution</td><td>HyClone</td><td>SH30042.01</td><td></td></tr><tr><td>45 &amp;mu;M cell strainer</td><td>VWR International Laboratory</td><td>195-2545</td><td></td></tr><tr><td>MediGel CPF with carprofen&amp;nbsp;</td><td>Controlled item from veterinary care in BCM</td><td></td><td>For pain management</td></tr><tr><td>Buprenorphine&amp;nbsp;</td><td>Controlled item from veterinary care in BCM</td><td></td><td>For pain management</td></tr><tr><td>Estradiol pellet</td><td>Innovative Research of America</td><td>SE-121</td><td></td></tr><tr><td>Ketamine and xylazine</td><td>Controlled item from veterinary care in BCM</td><td></td><td></td></tr><tr><td>Vet ointment</td><td>Controlled item from veterinary care in BCM</td><td></td><td>Avoid eye dryness</td></tr><tr><td>Shaver</td><td>Oster</td><td>78005-050</td><td>For furred mice</td></tr><tr><td>Isopropyl ethanol</td><td>ACROS</td><td>67-63-0</td><td></td></tr><tr><td>Betadine surgical scrub</td><td>Controlled item from veterinary care in BCM</td><td></td><td></td></tr><tr><td>#10 scalpel blades</td><td>Ted Pella, Inc</td><td>549-3CS-10</td><td>Multiple</td></tr><tr><td>No. 3 handle</td><td>Ted Pella, Inc</td><td>541-31</td><td>Need to be autoclaved</td></tr><tr><td>Sterile surgical drape</td><td>Sai Infusion Technology</td><td>PSS-SD1</td><td></td></tr><tr><td>Straight forceps&amp;nbsp;</td><td>Roboz Surgical Instrument</td><td>RS-5132</td><td>Need to be autoclaved</td></tr><tr><td>Straight fine forceps</td><td>Fine Science Tools</td><td>11253-20</td><td>Need to be autoclaved</td></tr><tr><td>Edged fine forceps</td><td>Fine Science Tools</td><td>11253-25</td><td>Need to be autoclaved</td></tr><tr><td>4-0 Vicryl silk suture</td><td>Johnson &amp;amp; Johnson Health Care</td><td>J214H</td><td></td></tr><tr><td>31 G insuline syringes</td><td>BD</td><td>328418</td><td>Multiple</td></tr><tr><td>Q-tips cotton swabs (Sterile)</td><td>VWR International Laboratory</td><td>89031-272</td><td></td></tr><tr><td>Skin glue</td><td>Henry Schein Animal Health</td><td>31477</td><td>For surgery site skin closure</td></tr><tr><td>Ear Tag Applicator</td><td>Fine Science Tools</td><td>24220-00</td><td></td></tr><tr><td>Ear tags</td><td>Fine Science Tools</td><td>24220-50</td><td></td></tr><tr><td>D-luciferin</td><td>Gold Biotechnology</td><td>LUCK</td><td>Avoid light and put on ice</td></tr><tr><td>28 G insulin syringes</td><td>BD</td><td>329410</td><td>For intra-orbital injection</td></tr><tr><td>Paraformadehyde</td><td>Alfa Aesar</td><td>30525-89-4</td><td>For tissue fixation</td></tr><tr><td>EDTA</td><td>OmniPur</td><td>4050</td><td>For bone tissue decalficication</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissection microscope</td><td>Leica</td><td>Leica S6E stereo</td><td></td></tr><tr><td>IVIS Lumina II imaging system</td><td>Advanced Molecular Vision</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-GFP antibodies (JL-8)</td><td>Clontech</td><td>632381</td><td></td></tr><tr><td>Anti-ALP antibodies</td><td>Abcam</td><td>ab108337</td><td></td></tr><tr><td>Anti-Osterix antibodies</td><td>Abcam</td><td>ab22552</td><td></td></tr></tbody></table>

intra-iliac artery injection for efficient and selective modeling of microscopic bone metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

그림 1은 해부학 적 위치와 총장 골 동맥 (적색)과 정맥 (파란색)의 관계를 보여줍니다. 그림 2는 해부 현미경 아래 장골 혈관과 신경의 상대적 위치를 보여줍니다. 도 2a에 도시 된 바와 같이, 혈관 신경 복막 벽 아래 오른쪽이고 피부 절개가 이루어지고, 복막 멀리 밀려 후에 밝혀 질 수있?...

Watch this Scientific Journal Video about Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis at JoVE.com

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

현미경 뼈 전이의 효율적이고 선택적 모델링을위한 내부 장골 동맥 주입

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

intra-iliac-artery-injection-for-efficient-selective-modeling

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

16.2K 조회수.  Baylor College of Medicine. This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types. 

비디오: Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

포탈 정맥 주입 모델 간 전이 유방암의 공부를

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

연구

암 연구학

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

자기 공명 영상, 체적 계산의 Tomography 및 초음파를 이용한 유방암의 뼈 전이의 누드 쥐 모델에서 Angiogenesis의 멀티 모달 이미징

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

의학

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

뼈 전이의 모델

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

두개 내 주입 및 자기 공명 이미징을 통해 뇌 전이를 모델링

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

원발성 뼈 종양 및 뼈에 이식된 고형 종양을 통한 뼈 전이 모델링

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

염증성 유방암 세포의 꼬리 정맥 주입을 통한 뇌 전이 모델링

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

경골 내 골육종 세포 주사 동위원소 골육종 및 폐 전이 마우스 모델 생성

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

암세포의 내경동맥 주입에 의한 뇌전이 모델링

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

컴퓨터 단층 촬영 및 골 형성 - 혈관 신생 커플 링의 광학 이미징 두개골 뼈자가 이식과 동종 이식 통합을 평가하기 위해

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

생체공학

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

인간 전립선암 세포의 심장내 주사를 통해 뼈 전이 이종이식 마우스 모델 생성

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...