Name: transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats
Uploaded: 2016-04-15T13:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.

주 : 다음 단계는 우리 기관의 지침과 바바리아, 독일의 지방 정부에 따라 수행되고있다. 지역 동물 관리 및 사용위원회에 의해 결정으로이 프로토콜을 재현하는 모든 시도는 동물의 인도적인 처리를위한 로컬 가이드 라인 준수에해야합니다. 예를 들어, 많은 기관은 멸균 장갑 설치류 수술 중 착용 할 것을 요구하고 있습니다. 또한, 바이러스와 작품은 개인과 환경 안전을위한주의하면서 현지 규정에 따라 수행해야합니다. 관리가 제대로 오염 된 폐기물 처분하고 (고온 가압 멸균 및 제조업체의 지침에 따라 적절한 항 바이러스 소독제로 청소하여, 예) 장비와 작업 공간을 청소주의해야한다. 시작 수술 전에 1. 준비 <ol><li> (고압 증기 멸균에 의해, 즉) 수술기구를 소독. </li><li> appropr에 바이러스를 희석사용되는 특정 바이러스의 독성 래트 균주의 민감성에 따라 달라질 늦었 농도. 주 : 수컷 버팔로 쥐 포성 구내염 바이러스를 사용하는 경우 예를 들어, 우리는 일반적으로 그 모델에서의 최대 허용 투여 량 인 1 ml의 부피에서 107 PFU를 주입. <ol><li> 1 ml를 주사기에 (무균 기술을 사용하여) 바이러스를 미리 기입하고, 주사기와 바늘에서 공기를 제거하기 위해주의하면서 30 G 바늘을 연결합니다. </li></ol></li><li> 필요한 모든 재료와 장비와 수술 공간을 준비하고 소독하는 에탄올로 영역을 스프레이. 거즈 면봉과 면봉 어플리케이터를 가습에 대한 멸균 생리 식염수 (0.9 % NaCl을)이있는 작은 용기를 입력합니다. </li></ol> 쥐 2. 준비 <ol><li> (로컬 동물 관리 및 사용위원회에 의해 지정된대로) 적절한 용량을 30 분에서 전에 수술 절차를 시작하기에, 진통제를 관리 할 수​​ 있습니다. 예를 들면,설피 린의 높은 용량 (100 ~ 200 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li><li> 흡입 (이소 플루 란) 또는 주사 (즉, 또한 MMF로 알려진 메데 토미의 혼합물 (0.15 ㎎ / ㎏), 미다 졸람 (2 ㎎ / ㎏)과 펜타닐 (0.005 ㎎ / ㎏)) 마취를 사용하여 쥐를 마취. 1-3%에 증발기를 통해 isolflurane 관리 정상 호흡 및 심박수를 유지하기 위해 필요한만큼 조정한다. <ol><li> 진행하기 전에, 쥐가 완전히 진정과 발 패드를 곤란하게하고 반응 관찰하여 통증을 느낄 수없는되어 있는지 확인합니다. </li><li> 마취 동안 건조를 방지하기 위해 동물의 눈 연고를 적용합니다. </li></ol></li><li> 단지 작은 동물 머리 깎기를 사용하여 성기 위 아래로 흉골에서 복부 영역을 면도. 피부를 부상없이 밀착 면도를 달성하기 위해주의하십시오. </li><li> 흡입 마취를 사용하여 적절한 크기의 코 콘에 주둥이를 배치하면, 앞장서고와 뒷다리에 테이프를 사용하여 수술 침대에 쥐를 수정합니다. 가열 패드를 배치래트 아래 체온을 유지한다. 방부제 페인트 알코올 린스 및 응용 하였다 외과 등급의 살균제를 사용하여 복부를 소독. </li></ol> 3. 개복술 <ol><li> 예 # 15로, 작은 만곡 메스를 사용하여, 직접 정중선을 따라 스킨 층의 작은 수직 절개를 만든다. xyphoid 과정까지 절개를 확장합니다. 다음 부드럽게 메스 절개를하기 Adson 집게 쌍으로 올려 근육층을 절개하고 수술 용 가위로 xyphoid 공정까지 절개를 연장한다. 복강의 장기를 천공하거나 xyphoid 프로세스 양쪽의 작은 혈관 손상되지 않도록주의한다. </li><li> 근육과 피부를 확산하고, 전체 복부를 노출 복부에 걸쳐 트랙터를 놓습니다. </li><li> 멸균 생리 식염수와 함께이 7.5 X 7.5 cm 2 거즈 면봉을 적셔 수술의 상단에 하나를 확산도 2a에 도시 된 바와 같이, 개방 및 하단에 하나. </li></ol> 간장 동맥 4. 분리 <ol><li> 부드럽게 복강 밖으로 장과 맹장을 들어 올려 하부 거즈 면봉에 배치, 멸균 생리 식염수에 적신이 젖은면 도포 면봉을 사용. 촉촉한 장기를 유지하고 길에서 그들을 유지하기 위에 거즈를 접으십시오. </li><li> 두 적신면 도포 면봉을 사용하여 부드럽게이 단계에서보기에 가장 지배적 인 로브 될 것입니다 왼쪽 측면 간 엽을 올립니다. 로브가 완전히 위쪽으로 이성을 상실하고 이전에 배치 거즈 면봉의 상단에 휴식을 할 수 있도록 로브의 밑면에있는 섬유 멤브레인을 통해 잘라 봄 가위의 작은 쌍을 사용하여 위쪽 로브 플립합니다. 제자리에 고정하기 위해 엽 위에 거즈 면봉을 접습니다. </li><li> 지금보기에서 가장 전엽 인 다음 로브 (전방 미상)를 계속합니다. 번째로해부 현미경 미세 겸자 E 보조제,이를 분리하고 자유롭게 위쪽으로 대칭되도록하기 위하여 로브를 둘러싼 박막을 해부. 왼쪽 측면 로브와 함께 거즈 면봉 아래 로브를 놓습니다. </li><li> 지금 볼 수 있습니다 후방 꼬리가 로브를 관찰합니다. 완전히 후방 꼬리가 로브를 해부 위쪽과 거즈 안에 함께 이전에 배치 간 로브와 그것을 뒤집어 단계를 반복 4.3. </li><li> 그냥 꼬리가 로브의 원래 위치 아래에, 지금 눈에 보이는 쉽고 강력한 맥동에 의해 인식 될 것입니다 일반적인 간동맥을 찾을 수 있습니다. 해부학 오른쪽에있는 동맥을 이어보기에서 십이지장과 적절한 간 동맥을 가리는 두꺼운 멤브레인 재료를 관찰합니다. 미세 집게를 사용하여이 물질을 들어 올려 조심스럽게이 지역에있는 작은 혈관을 피하기 위해주의하면서 그것을 찢어. 발생할 수있는 출혈을 멈추게하는면 도포 면봉을 사용합니다. <ol><li> ㄱ을 찾습니다멤브레인이 제대로 해부하면 해부 현미경의 도움을 명확히 볼 수 rterial 분기. </li></ol></li><li> 이 좋은, 무 외상성 집게를 사용하여 조심스럽게 일반적인 간 동맥, 위 십이지장 동맥, 그들은 주변의 막 (그림 2B)에서 해방되도록 적절한 간 동맥을 해부하다. </li></ol> 주입을위한 동맥 5. 준비 <ol><li> 십이지장 동맥 아래에 7-0 prolene의 봉합을 전달, 그냥 분기 (적절한 배치를위한 그림 1 참조) 이상, 선단에 영구적 인 끈을 묶어 마이크로 바늘 홀더를 사용합니다. 끝 부분에 남아있는 봉합 재료의 약 2 인치를 허용하고도 2c에서와 같이 가르쳐 동맥을 당겨 바늘 홀더를 고정. </li><li> 일시적으로 혈액의 흐름 (그림 2C)를 차단하는 일반적인 간 동맥에 작은 클램프를 놓습니다. 참고 :이 꼬마 도깨비입니다바늘이 혈관으로부터 제거되면 ortant 다량 출혈을 방지한다. 또한, 동맥 혈류의 차단은 느린 바이러스 관리 및 더 나은 전달 효율성을 수 있습니다. <ol><li> 중요 혈액 흐름이 정지되는 시간 제한 신속하게 작업 - 공통 간동맥 클램핑 5 분 이내에 동맥 주입 이후 결찰을 수행한다. </li></ol></li><li> 가능하다면, 위 십이지장 동맥 날카로운 초점이 정확하게 30 G 바늘을 삽입하기에 충분히 큰 표시되도록, 해부 현미경의 배율을 높일 수 있습니다. 십이지장 동맥의 내강에 주입 주사기의 바늘을 놓고 플런저를 밀어 천천히. 참고 : 바늘의 적절한 배치는 적절한 간동맥을 통해 간으로 직접 흐르는 주입 된 용액에 발생합니다. <ol><li> 전달 효율을 최대화하는 것이 매우 천천히 주입을 수행한다. </li></ol></li> &lt;리&gt; 전체 주입량을 투여 한 후, 천천히 동맥 바늘 후퇴. 참고 : 주사 부위로부터 출혈이 없어야합니다. 출혈이 발생 않는 경우에는, 상기 클램프가 적절하게 배치되지 않고 신속하게 조정되어야 함을 나타낸다. </li></ol> 6. 결산 <ol><li> 십이지장 동맥 아래 두 번째 7-0 prolene의 봉합을 전달하고, 주사 부위 위에 제 2 영구 합자 넥타이. </li><li> 일반적인 간동맥에서 클램프를 제거합니다. 간으로 적절한 간 동맥을 통해 혈액의 흐름을 확인하는 복원되었습니다. <ol><li> 합자의 느슨한 끝을 잘라. </li></ol></li><li> 복강에 창자와 간을 반환하기 전에 더 출혈이 없음을 확인합니다. 부록은 원래의 방향으로 반환주의하십시오. </li><li> 곡선 바늘로 4-0 vicryl 봉합사 재료를 사용하여 별도의 레이어에서 근육과 피부를 봉합. 장소 continuou의 봉합은 약 1mm 떨어져 단단히 잡아 당깁니다. <ol><li> 봉합 단계에서, 진통제 등의 부 프레 노르 핀 등 (0.05 ㎎ / ㎏)을 관리 할 수​​ 있습니다. </li></ol></li><li> 마취에서 쥐를 제거합니다. 이소 플루 란이 사용 된 경우, 래트는 몇 분 안에 의식을 회복한다. MMF는 주사 마취로 사용 된 경우, 마취 효과를 반전되는, Atipamezole (0.75 ㎎ / ㎏), 플루 마제 닐 (0.2 ㎎ / ㎏) 및 날록손 (9.12 ㎎ / ㎏)의 피하 주사로 길항 과 동물이 20 분 안에 의식을 회복 할 수 있습니다. 초기 각성 단계에서 적외선 온난화 램프와 따뜻한 쥐를 유지합니다. <ol><li> 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. </li><li> 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. </li></ol></li><li> 고통의 병적 징후가 수술 후 쥐를 모니터링하고 follow-진통제와 로컬 동물 관리 및 사용위원회가 필요. 예를 들면, 부 프레 노르 핀은 / 0.05 mg을 투여 12 시간마다 kg을 할 수있다. </li></ol>

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The authors have nothing to disclose.

Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to ...

간세포 암 (HCC)은 전 세계적으로 다섯 번째 가장 일반적인 암, 그리고 중요한 건강 문제 1,2하고 암 관련 사망의 세 번째 주요 원인이다. 색전술 (TAE) 또는 화학 색전술 (TACE)을 포함하는 종양 절제술, 또는 그 기다리고 간 이식, 국소 치료를받을 수없는 환자의 표준 완화 의료 3,4으로 적용됩니다. 주변 간 간문맥 5,6-로부터 혈액 공급의 대부분을 수신하면서 이들 치료법은 종양 간 동맥 혈류가 거의 독점적으로 공급되고, 이에 의해 간에서 이중 혈액 공급의 고유 기능을 이용한다. HCC에 대한 설립 치료의 매우 제한된 효능으로 인해 종양 살상 바이러스는 유망한 대체 치료제로 등장했다. JX-594는 최근 Pexa-VEC 이름을 변경하는 티미 딘 키나제 삭제 된 우두 벡터, 과립구-m로 무장이다HCC 7의 II 상 임상 시험이 완료된 acrophage 콜로니 자극 인자 (GM-CSF). 최근 인간 인터페론 - 베타를 발현하는 재조합 수 포성 구내염 바이러스 벡터 (VSV)는 sorafenib-내화 HCC (NCT01628640)에 대한 상 임상 시험을 체결했다. 종양 살상 바이러스가 가까운 환자에서 간세포 암종에 대한 임상 응용 프로그램에 대한 승인을 얻기로 이동함에 따라, 효과적인 투여 경로에 대한 필요성은 다 초점 질병이 분명하다 목표로한다. 전신적 전달로 인해 비효율적 종양 전달에 크게 효과가 있지만, 애플리케이션은 종양 질환 진행에 민감 uninjectable 미세한 병변을 떠나는 주입 종양 치료의 효능을 제한 할 수있다. 우리는 소성을 HCC를 대상으로하는 국소 방식으로 콜리 틱 바이러스 치료를 관리하는 쥐의 간 동맥을 분리하는 방법을 설립했다. 우리는 증명이 안전하고 효과적인 광고에서이 투여 경로 결과면역 능력이 쥐 8-10에서 중요한 생존 연장의 결과로, 다 초점 HCC 결절의 전달을했습니다. 여기서는, 액세스 해부 및 쥐의 간동맥에 주입하는 방법을 설명한다. 절차의 방식은도 1에 도시된다 (이전 9 공개)

<table><tbody><tr><td>Veterinary clippers</td><td>Aesculap</td><td>GT415</td><td>Small, cordless trimmer ideal for removing fur from surgical area</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>30 G Needles</td><td>Braun</td><td>4656300</td><td>30 G x &amp;frac12;&amp;rdquo;</td></tr><tr><td>1 ml syringes</td><td>Braun</td><td>9161406V</td><td>Tuberculin syringe</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#15</td><td>#15 blade&amp;nbsp;</td></tr><tr><td>Standard surgical scissors</td><td>Fine Science Tools</td><td>14001-13&amp;nbsp;</td><td>Sharp/blunt, for opening skin and muscle</td></tr><tr><td>Adson forcep</td><td>Fine Science Tools</td><td>1101-12</td><td>With teeth, for grasping skin and muscle</td></tr><tr><td>Alm retractor</td><td>Fine Science Tools</td><td>17008-07</td><td>With blunt teeth, for spreading abdominal cavity open during surgery</td></tr><tr><td>Gauze swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>18504</td><td>7.5 cm x 7.5 cm, should be autoclaved prior to use</td></tr><tr><td>Cotton-tipped applicator swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>11970</td><td>Sterile</td></tr><tr><td>Fine-tipped foreceps</td><td>Fine Science Tools</td><td>11063-07</td><td>0.4 mm, angled tip, for dissecting hepatic artery</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>For delicate cutting</td></tr><tr><td>Micro-needle holder</td><td>Fine Science Tools</td><td>12076-12</td><td>For ligating gastroduodenal artery</td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12005-15</td><td>Tungsten carbide jaws</td></tr><tr><td>7-0 Prolene sutures</td><td>Ethicon</td><td>8648H</td><td>Polypropylene suture with curved needle, for ligating gastroduodenal artery</td></tr><tr><td>4-0 Vicryl sutures</td><td>Ethicon</td><td>V3040H</td><td>With curved needle attached</td></tr><tr><td>Infrared warming lamp</td><td>Beurer</td><td>IL11</td><td>For maintaining body temperature post-operatively</td></tr></tbody></table>

transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

경험 (동맥 성공적 해부 주입 한 동물이 수술을 살아되었음을 의미 함)이 거의 100 % 성공률을 달성 할 수있다. 그러나, (기타 종양 부담 정도 기본적인 간 기능) 수술 전에 래트의 상태 분명 수술 개입의 결과에 중요한 역할을한다. 수술 후, 래트를 단독 버퍼해도 바이러스없이 주입하고, 일시적인 체중 감량과 간 효소의 약간의 일시적인 상승이 발생할 것으로 예상 ?...

Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

쥐에서 동소 이식 간세포 암의 국소 치료에 대한 종양 살상 바이러스의 간동맥 관리

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

transarterial-administration-oncolytic-viruses-for-locoregional

Research

JoVE Journal

Medicine

9.4K 조회수.  Klinikum rechts der Isar der TU München. Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

비디오: Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

재조합 Adeno - 관련 바이러스 혈청형 3 벡터에 의해 간암 세포의 고효율 전달

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

연구

의학

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

쥐에게 Orthotopic 간 이식

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

간 간선 재건과 부분 Orthotopic 간 이식의 쥐 모델에 대한 외과 절차

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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 ...

암 연구학

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

마우스에서 연속 국소 키메라 항원 수용체(CAR) T 세포 주입을 위한 두개내 캐뉼라 이식

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

종양 살상 바이러스의 전 임상 평가에 대한 연구에면 쥐의 취급

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

메서드 및 Adeno 관련 바이러스를 쥐의 정 맥 관리 및 중앙 신경 전달의 평가 대 한 팁

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

신경과학

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

다시는 vivo에서 선박 지향 70% 쥐 부분 간 이식 Arterialized Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

Orthotopic의 쥐 모델 간 이식 Suprahepatic 베 나 정 맥 재건에 대 한 새로운 자석 문 합 기술을 사용 하 여

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

직교 간 이식의 쥐 모형에 있는 동맥 재연결 후에 감소된 합병증

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...