The authors have no acknowledgements.

윤리 문 : 모든 동물 연구는 텍사스 MD 앤더슨 암 센터의 대학 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 사출에 대한 암 세포를 준비 <ol><li> 주입 이전에 암 세포를 하루나 이틀 종자. 전문 매체가 특정 세포주에 대한 문헌에 표시되지 않는 한, 10 % FBS로 보충 된 DMEM / F12 미디어를 사용합니다. </li><li> 37 ℃에서 2 분 - 1 트립신 (0.25 %) 이전에 한번 혈청이없는 배지로 세척하여 처음 80 % 합류 - 수술 당일 수확 세포들은 70에 도달 할 때. DMEM / F12 배지를 FBS 함유 10 %는 0.25 % 트립신을 종결 세포에 추가한다. </li><li> 3 분 동안 80 × g에서 원심 분리하여 세포. 세포주에 따라, 5 × 106 세포 / ml - 잔류 혈청을 제거 혈구와 세포를 세어 1 행크의 균형 잡힌 염 용액 (HBSS)에 재현 탁 두번 무 혈청 배지에서 세포를 세척 하였다. 얼음에 보관주입 때까지. 주 : MDA-MB-231 유방암 세포 K1735 흑색 종 세포는이 연구에 사용되었고, 두 세포주 / ml의 농도를 2 × 106 세포로 사용 하였다. 전이 분석의 결과에 영향을 미칠 수 overtreatment 최대한 짧게 트립신 처리 시간을 유지한다. </li></ol> 2. 종양 세포 주입을 위해 마우스를 준비합니다 <ol><li> 복강 내 (IP)에 의해 마우스 케타민의 주입 / 자일 라진 칵테일 (케타민 100 ㎎ / ㎏, 자일 라진 10 ㎎ / ㎏)를 마취. </li><li> 동물의 발을 곤란하게하여 완전한 마취를 확인하고 응답의 부족을 관찰합니다. </li><li> (단백질 젤 주조에 사용) 유리 접시에 마우스를 놓고 고무 밴드로 고정합니다. </li><li> 필요한 경우, 면도 또는 털 제거 제품을 소량 도포하고 종이 타월로 머리를 닦아 목 모발 제거. 참고 : 누드 마우스를 사용하는 경우가 체모를 가지고 있지 않는 한,이 단계를 건너 뜁니다. </li><li> 적용하여 목 피부를 청소포비돈 요오드 및 70 % 알콜. </li><li> 해부 현미경의 무대에 마우스를 놓습니다 </li><li> 긴 수술 메스로 피부 ~ 1cm의 절개를합니다. </li><li> 퉁명스럽게 아래에있는 경동맥을 노출 이빨 집게로 근육을 해부하다. </li><li> 수술 집게와 인접 미주 신경에서 경동맥을 분리합니다. </li><li> 수술 집게를 사용하여 작은 목화 공에, 목화 공에서 어떤면을 분리 축축하고, 패션. 주사 원하는 사이트의 경동맥 아래에이 버퍼 수분 목화 공을 놓습니다. </li><li> 코튼 볼에 봉합 원위부 및 근위부를 놓고 느슨한 매듭을합니다. 주사 부위에 혈류를 차단하는 근위 매듭을 조인다. </li><li> 면화 공 경동맥 신선한 붉은 피와 완벽하게 가압 나타나는 경우 암 세포 주입을 진행합니다. </li></ol> 경동맥에 3. 암 세포 주입 <ol><li> 소용돌이와 100 μL O를 그립니다주사기로 F 암세포. </li><li> 해부 현미경, 천천히면 공의 꼭대기에 앉아 경동맥의 루멘에 (경사 최대) 31 G 바늘을 삽입합니다. </li><li> 천천히 경동맥 내로 주사기로부터 세포를 주입. 버퍼 암 세포가 순환으로 가압 될 때 사출 성공적인 인근 혈관 근육의 색을 변경하여 현미경으로 관찰 할 수있다. </li><li> 전체 볼륨의 주입이 완료되면, 역류 방지 부드럽게 원위 경동맥 리프트. </li><li> 빠르게 주입 프로세스를 완료 원위 매듭을 조인다. </li><li> 주입을 완료 한 후, 합자을 강화하고, 해부 현미경 하에서 미세 가위로 과잉 실크 봉합사 트림. 상처 부위를 덮도록 분리 근육 위로 이동한다. 두 스테이플 피부를 닫습니다. </li></ol> 4. 수술 복구 <ol><li> 가열 패드로 조작 된 동물 이동복구. 동물이 의식을 회복하는 데 60 분 - 그것은 30 걸릴 수 있습니다. 한편, 수술 후 통증 등의 피하 주사를 통해 프레 노르 핀 (0.05 ㎎ / ㎏)을 관리 할 수 있습니다. 참고 : 진통은 해당 지역의 IACUC 프로토콜을 승인에 따라 제공되어야한다. </li><li> 동물 각성과 정상적인 동작을 재개하면, 자신의 주택 위치에 마우스를 반환합니다. 며칠 후 수술 젤 음식 마우스를 제공합니다. 수술 기록을 유지하고 실내에 보관합니다. </li></ol> 생체 이미징 5. 비 침습적 <ol><li> 루시퍼 라제 리포터 작 제물로 표지 세포, 생체 내 이미징 시스템 (14)에 사용하여 암세포 주입 효율을 측정한다. 주 : 이미징 연구 동물에게 과도한 스트레스를 방지하기 위해 주입 당일에 수행되지 않는다. </li></ol>

<ol>
	<li>Gavrilovic, I. T., Posner, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metastases:+epidemiology+and+pathophysiology.">Brain metastases: epidemiology and pathophysiology.</a> J Neurooncol. 75 (1), 5-14 (2005).</li><li>Patchell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+management+of+brain+metastases.">The management of brain metastases.</a> Cancer Treat Rev. 29 (6), 533-540 (2003).</li><li>Barnholtz-Sloan, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+proportions+of+brain+metastases+in+patients+diagnosed+(1973+to+2001)+in+the+Metropolitan+Detroit+Cancer+Surveillance+System.">Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the Metropolitan Detroit Cancer Surveillance System.</a> J Clin Oncol. 22 (14), 2865-2872 (2004).</li><li>Schouten, L. J., Rutten, J., Huveneers, H. A., Twijnstra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94 (10), 2698-2705 (2002).</li><li>Bendell, J. C., 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=Central+nervous+system+metastases+in+women+who+receive+trastuzumab-based+therapy+for+metastatic+breast+carcinoma.">Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma.</a> Cancer. 97 (12), 2972-2977 (2003).</li><li>Clayton, A. 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=Incidence+of+cerebral+metastases+in+patients+treated+with+trastuzumab+for+metastatic+breast+cancer.">Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer.</a> Br J Cancer. 91 (4), 639-643 (2004).</li><li>Romond, E. 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=Trastuzumab+plus+adjuvant+chemotherapy+for+operable+HER2-positive+breast+cancer.">Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer.</a> N Engl J Med. 353 (16), 1673-1684 (2005).</li><li>Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patient+perspective+on+brain+metastases+in+breast+cancer.">A patient perspective on brain metastases in breast cancer.</a> Clin Cancer Res. 13 (6), 1623-1624 (2007).</li><li>Lin, N. U., Bellon, J. R., Winer, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J Clin Oncol. 22 (17), 3608-3617 (2004).</li><li>Zhang, C., Yu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment+determinants+of+brain+metastasis+of+brain+metastasis.">Microenvironment determinants of brain metastasis of brain metastasis.</a> Cell Biosci. 1 (1), 8 (2011).</li><li>Fidler, I. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+selectivity+for+implantation+survival+and+growth+of+B16+melanoma+variant+tumor+lines.">Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines.</a> J Natl Cancer Inst. 57 (5), 1199-1202 (1976).</li><li>Zhang, 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=Microenvironment-induced+PTEN+loss+by+exosomal+microRNA+primes+brain+metastasis+outgrowth.">Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth.</a> Nature. 527 (7576), 100-104 (2015).</li><li>Zhang, 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=SRC+family+kinases+as+novel+therapeutic+targets+to+treat+breast+cancer+brain+metastases.">SRC family kinases as novel therapeutic targets to treat breast cancer brain metastases.</a> Cancer Res. 73 (18), 5764-5774 (2013).</li><li>Lim, E., Modi, K. D., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescent+imaging+of+mammary+tumors+using+IVIS+spectrum.">In vivo bioluminescent imaging of mammary tumors using IVIS spectrum.</a> J Vis Exp. (26), (2009).</li><li>Zhang, C., Zhang, F., Tsan, 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=Transforming+growth+factor-beta2+is+a+molecular+determinant+for+site-specific+melanoma+metastasis+in+the+brain.">Transforming growth factor-beta2 is a molecular determinant for site-specific melanoma metastasis in the brain.</a> Cancer Res. 69 (3), 828-835 (2009).</li></ol>

The authors have nothing to disclose.

암 세포의 성공적인 경동맥 동맥 주입을위한 가장 중요한 단계는 다음과 같습니다 수술 준비 마우스의 1) 깊은 마취; 2)면 지원의 상단에 경동맥의 정상 위치; 성공적인 주사 후 경동맥의 3) 단단히 결찰. 30 분 ~ 깊은 마취는 해부 현미경으로 꾸준한 수술 성능을 위해 일반적으로 필요하다. 우리는 마우스 마취 상업적으로 즉시 사용 케타민 / 자일 라진 칵테일을 사용합니다. 마...

중추 신경계 (CNS)에 암의 전이는 파괴적인 질환, 뇌 실질 또는 ( &quot;뇌 전이는&quot;이 문서에 모두를 의미)를 leptomeninges를 포함 할 수있다. 1 일, 2 : 그것은&gt; 10 차 신경 교종을 상회, 주된 두개 내 악성 종양이다. 폐암, 유방암, 흑색 종 및 뇌 전이 3,4- 높은 빈도를 생성하는 세 가지 주요 종양 질환이다. 최근, 새로운 암 치료의 인상적인 발전이 크게 많은 암 환자의 생존 및 삶의 질 개선을 연장하고있다. 그러나, 재발시 뇌 전이의 빈도가 빠르게 증가하고있다. 예를 들어, 항 HER2 항체 트라 스투 주맵 (헤르 셉틴)은 HER2 + 유방암 환자에서 심각한 임상 적 효능을 보여 주었다; 아직 불안 추세가 등장했습니다이러한 환자 : 그 두개 외 전신 질환 초기에 나중에 뇌 전이 5, 6, 7을 개발 트라 스투 주맙 치료 혜택을 사람들의 1/3까지. 슬프게도, 뇌 전이 환자는 일반적으로 삶의 질 외상 저하를 경험하고, 거의 모든 현재의 치료에 불응하고, 진단 후 자신의 일 년 생존율은 ~ 20 % 8입니다. 현재 (스테로이드, 두개골 방사선 치료, 선택된 환자에서 수술 적 절제 포함) 뇌 전이에 대한 치료입니다 단지 9 치료하지 완화. 따라서, 뇌 전이는 새로운 암 치료의 시대에서 다음 당당한 도전으로 부상하고있다. 환자가 병원에 ​​매일 직면 충족 문제를 해결하기 위해, 우리는 긴급하게 더 나은 뇌 전이의 기본 메커니즘을 이해하고 새로운 치료법을 개발하기 위해이 지식을 사용해야합니다. <p클래스 = &quot;jove_content&quot;&gt; 전이성 암 세포에 의한 뇌의 성공적인 정착은 혈액 뇌 장벽 (BBB)의 바이 패스 여러 생물학적 플레이어의 복잡한 상호 작용에 의해 매개되는 기능의 시리즈의 성능을 요구하고 뇌의 극한 탈출 생체 외 또는 시험 관내 시스템에서 효과적으로 요약되어 어느 것도 면역 방어 메카니즘 (10). 따라서, 생체 내 모델에서 적절하고 충실한 뇌 전이의 연구에 중요하다. 생체 전이 분석에서 종래의 폐에 박혀 될 셀의 대부분을 이끌어 꼬리 정맥 주사를 통해 암세포를 도입한다. 동물의 죽음은 폐 (11)에 종양 부담에 의해 발생하기 전에 뇌 전이 병변은 거의 이러한 모델에서 생산되지 않습니다. 암세포를 직접 뇌내 주입은 CNS에서 일관된 종양 생장을 생성 널리 차 신경 교종의 연구에 사용된다. 그러나이야UCH 주입이 모델의 생리 학적 관련성에 관해서, 주요 관심 사항 모두를 BBB 타협 및 주사 부위에서 외상시킨다. 또 다른 자주 사용되는 암세포 도입 경로, 심장 내 주사, 관리하고 CNS로 실험 전이를 생성 않기 쉽다. 그러나, CNS 이외 장기 사이트에 대한 동시 전이가 항상 일어난다 동물 11 사망을 일으킬 수있다 따라서,이 모델의 변동의 높은 생물학적 메커니즘이나 동물의 제한된 수의 치료제의 정량 평가가 부적절한다. 여기서 우리는 경동맥에 암세포를 주입 실험을 통해 뇌 전이를 생성하는 절차를 설명한다. 우리는 뇌 전이의 전이성 캐스케이드 개별 유전자 &#39;기여를 해부하고 치료 개입의 효과를 평가하기 위해이 방법을 사용했다S (12, 13). 이 방법의 주요 이점은 재현성 및 변동의 낮은 정도의 높은 수준이다; 주요 단점은 미세 수술을 수행하기 위해 필요한 정교함 손재주이다.

<table border="0" cellpadding="0" cellspacing="0" width="1156">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Ketamine hydrochloride/xylazine hydrochloride solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:151px;">K113</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Routine Stereomicroscope Leica M50</td>
			<td style="width:151px;">Leica</td>
			<td style="width:151px;">M50</td>
			<td>The microscope is modular and highly configurable to fit particular space requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Surgical disposable scapel</td>
			<td style="width:151px;">Integra Miltex</td>
			<td style="width:151px;">4-410</td>
			<td>to make skin incisions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tissue Forceps - 1x2 Teeth</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11021-12</td>
			<td>to bluntly dissect mucles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dumont #5 - Mirror Finish Forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11252-23</td>
			<td>to separate and prepare the carotid artery for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">15025-10</td>
			<td>to cut sutures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">EZ Clip Kit</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:151px;">59020</td>
			<td>for wound cloure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BD Insulin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:151px;">328438</td>
			<td>for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">IVIS Spectrum In Vivo Imaging System</td>
			<td style="width:151px;">PerkinElmer</td>
			<td style="width:151px;">124262</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracarotid cancer cell injection to produce mouse models of brain metastasis

전이, 환자의 신체 내에서 보조 사이트의 확산과 악성 세포의 성장, 암 관련 사망의&gt; 90 %를 차지한다. 최근, 새로운 치료의 인상적인 발전은 극적으로 많은 암 환자의 생존과 삶의 품질 향상을 연장했다. 슬프게도, 뇌 전이 재발의 빈도는 빠르게 상승하고, 현재의 모든 치료는 단지 완화 있습니다. 따라서, 좋은 실험 동물 모델이 긴급하게 질병 생물학에 대한 심층 연구를 촉진하고 전임상 평가를위한 새로운 치료 요법을 평가하기 위해 필요하다. 그러나 암세포의 꼬리 정맥 주사를 통하여 생체 전이 분석에서 표준은 주로 폐 전이성 병변을 생성한다; 동물은 일반적으로 뇌 전이의 의미를 가지 전에 폐 종양 부담에 굴복. 종양 세포의 주입 심내 뇌를 포함한 여러 기관 사이트 전이성 병변을 생성; 그러나 variabi이 모델로 제작 종양 성장의 lity는 치료 효과를 평가하는 자사의 유틸리티를 완충, 큽니다. 뇌 전이의 연구를위한 안정적이고 일관성있는 동물 모델을 생성하려면, 여기에 우리가 종양 세포의 intracarotid 주입을 통해 집 마우스 (뮤스의 musculus)에서 실험적인 뇌 전이를 생성하는 절차를 설명합니다. 이러한 접근 방식은 기본 따라서 생물학적 메커니즘을 연구 및 신규 한 치료제를 평가하는 연구 노력이 용이 한 유사 성장과 사망률 특성 뇌 전이 - 함유 마우스의 다수를 생성 할 수있다.

분사의 품질이 평가 될 수있는 두 지점이있다. 첫 번째 가능성은 주입 중에 혈관 색을 변경하는 운전자의 관찰이다. 불량한 주사 누설 손쉽게 해부 현미경으로 관찰 할 수있다. 정상 및 보안 마우스 본체의 위치 (그림 1A)과지지면 공의 경동맥 (그림 1B)는 경동맥에 원활하고 성공적인 주입을위한 중요한 요소입니다. 주입 품질을 평가하기위한 두번...

Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis at JoVE.com

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

치료 옵션을 완화 남아있는 동안 그 빈도는 증가로 뇌 전이는 긴급 충족되지 않은 의료 요구되고있다. 암 세포의 intracarotid 동맥 주입을 통해 뇌 전이의 실험 동물 모델을 만들기 소설 개입 요법의 질병 생물학 및 평가의 기계론의 연구를 용이하게한다.

Intracarotid 암 세포 주입 뇌 전이의 마우스 모델을 제작하기

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

intracarotid-cancer-cell-injection-to-produce-mouse-models-brain

Research

JoVE Journal

Cancer Research

26.2K 조회수.  The University of Texas MD Anderson Cancer Center.  치료 옵션을 완화 남아있는 동안 그 빈도는 증가로 뇌 전이는 긴급 충족되지 않은 의료 요구되고있다. 암 세포의 intracarotid 동맥 주입을 통해 뇌 전이의 실험 동물 모델을 만들기 소설 개입 요법의 질병 생물학 및 평가의 기계론의 연구를 용이하게한다. 

비디오: Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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.

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 ...

연구

암 연구학

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

전임상 마우스 모델에 대한 수술 절차 및 방법론<em&gt; 드 노보</em&gt; 유방암 전이

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

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 ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

반대로 mitotic 에이전트 Docetaxel에 전립선 암 세포 모델의 생성

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

라이브 셀 이미징 분석 실험 연구 세포종 뇌 종양 줄기 세포 이동과 내 습을

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

인간 비뇨기과 세포 암 및 대장암 종양 성장 및 자발적 전이를 위한 환자 유래 직교 이종이식 모델

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

종양 세포의 측면 꼬리 정맥 주입 다음 폐 전이의 병리학 적 분석

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

중추신경계 전이의 환자 유래 이종이식 모델의 확립 및 활용

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

간 전이 스트로마를 연구하기 위해 대장암 오가노이드의 포털 정맥 주사

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...