Name: an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis
Uploaded: 2019-09-12T13:00:00
Description: Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

이 작품은 NIH / NCI R01 CA16435-01A1 (K. F. 스타벨리 - 오 캐롤, PI) 및 NIH / NCI R01CA208396 (마크 케스터, 광푸 리, 케빈 F. 스타벨리 - 오 캐롤)에 의해 지원됩니다.

참고: 동물 과목을 포함한 모든 절차는 미주리 대학의 IACUC에 의해 승인되었습니다. 모든 마우스는 "실험실 동물의 관리 및 사용에 대한 가이드"에 설명 된 기준에 따라 인도적 치료를 받았다. 세포 격리 및 예방 접종에 대한 다음 절차는 후드에서 수행해야합니다. 모든 연주자는 마우스와 조직을 처리하기 위한 표준 개인 보호 장비를 착용해야 합니다.
1. 탄소 사염화물의 IP 주?...

<ol>
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이 프로토콜을 통해 우리는 인간 HCC 개시 및 진행을 모방하는 HCC의 신뢰할 수 있고 재현 가능한 뮤린 모델을 확립했습니다. 임상적으로, 많은 위험 요소는 연속적으로 간 손상을 유도, 간 섬유증, 간경변과 HCC의 최종 단계. 우리의 프로토콜에서, CCl4의 IP 주입은 먼저 야생 형 마우스에서 간 섬유증을 생산하는 데 사용되며, 이는 후속 종양 발생 간 세포가 간 섬유증의 설정에서 종양을 형성 ...

간세포암(HCC)은 미국에서 가장 빠르게 증가하는 유형의 암(미국)1,2,3. 매년 약 850,000개의 새로운 사례가4,5 및 700,000명의 환자가 이 치명적인질병으로6, 7,8,9,10으로 진단됩니다. 전 세계적으로 암 관련 사망의 두 번째로 높은 원인입니다. HCC의 관리는 외과적 절제술, 이식, 절제, 화학색전증, 또는소라페닙(sorafenib) 과같은 전신 요법을 포함한다. 외과 절제술 또는 이식으로 조기 진단 및 관리는 전체 생존 율이 가장 높은4. 불행하게도, 환자의 대다수는 나중 단계에서 나타나고 절제, 화학색전증 또는 sorafenib12를가진 관리를 요구합니다. 소라페닙, 수용 체 티로신 키 나 제 억제제 (RTKI), 에 식품 의약품 안전 청에 의해 승인 되었다 2008 절제 할 수 없는 HCC 치료에 사용할 수 있는 유일한 전신 약물 치료로. 약물은 전체 생존율이 7.9개월에서10.7개월13개월로소폭 증가하지만, HCC 관리에 활용될 수 있는 새로운 치료 전략을 제공했다.
확립된 암을 제거하기 위해 면역체계를 조작하는 것은 암 연구에서 급속히 성장하고 있는 분야이다14. 면역 검사점 연구는 암 치료15,16에서면역 치료 약물 개발을 상당히 진전하였다. FDA는 세포독성 T-림프구 항원 4(CTLA-4), 프로그램된 세포 사멸 단백질 1(PD-1), 흑색종, 폐암, 두경부암 및 방광암 치료를 위한 리간드 PD-L1에 대한 항체(Abs) 사용을승인했다. 18세 , 19세 , 20.진행 중인 HCC의 치료를 위해 PD-1, PD-L1 또는 CTLA-4에 대하여 하나 또는 다중 항체를 이용한 단독요법 또는 병용 요법의 임상 시험이 진행 중이다21,22,23,일부 시험은 유리한 결과를 보여 주었다. 2017년, FDA는 소아페닙에 대한 내성이 있는 HCC 환자를 치료하기 위해 항 PD-1 항체에 대한 조기 승인을 승인했지만, 이 치료법의 전반적인 반응률은 14.3%에 불과합니다. 다른 전략은 이 때 임상 실습으로 번역되지 않았습니다24,25. 면역체크체크치료개선을위한종양유발심오한면역내성을극복26; 면역 체크포인트 치료의 효능을 예측; 면역 관련 부작용 방지; 최적화 관리 경로, 복용량, 그리고 주파수; 그리고 치료의 효과적인 조합을 찾는27,28,29 모두 매우 어려운 작업 남아있다.
현재 마우스 모델에서 HCC를 유도하는 데 사용되는 몇 가지 통상적인 접근법이 있으며, 조사자의 특정 연구 질문30에따라 활용되고 있다. 화학적으로 유도된 HCC 마우스 모델은 유해성 화합물을 가지고 상해 유발 악성을 모방한다. HCC 세포주의 자궁외 또는 직교 이식을 통한 이종이식 모델은 약물 스크리닝에 적합합니다. 많은 유전자 변형 마우스가 HCC의 병리생리학을 조사하도록 설계되었습니다. 바이러스 성 유전자를 발현 하는 형질 전환 마우스, 종양 유전자 및 성장 인자 간 암 선 에서 관련 된 경로의 식별을 허용. 본래의 한계로 인해, 이러한 모델은 인간 HCC에서 볼 수 있는 전형적인 면역 특성을 되풀이하지 않으며, 이는 근본적인 메커니즘의 해명과 혁신적인 면역 치료 전략의 개발을 크게 저해했습니다14 ,15. 우리는 최근에 임상적으로 관련있는 뮤린 모형을 만들었습니다. 이 새로운 모델은 인간 HCC 개시 및 진행을 모방할 뿐만 아니라 면역 기능 장애를 포함한 인간 질병의 가장 전형적인 특징을 반영합니다. 우리는 그것의 생물학적 및 면역학적 특성을 특징으로 합니다. 이 새로운 모델을 활용하여 HCC31, 32,33,34,35,36을치료하기 위한 다양한 면역 치료 전략을 탐구했습니다. 37. 이 독특한 플랫폼은 종양 유발 면역 내성의 메커니즘을 연구하고 최종 임상 번역을 향한 HCC에 대한 개념 증명 치료 전략을 개발할 수 있게 합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Anesthesia machine</td>
			<td style="width:123px;">VETEQUIP</td>
			<td style="width:344px;">IMPAC6</td>
			<td style="width:167px;">anesthesia machine for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle</td>
			<td>BD</td>
			<td>8122963</td>
			<td>Needle used for liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Jackson Lab</td>
			<td>000664&#160;</td>
			<td>mice used in prototol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>CRESCENT CHEMICAL</td>
			<td>20402</td>
			<td>carprofen for pain release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer&#160;</td>
			<td>CORNING</td>
			<td>REF 431751</td>
			<td>Cell strainer, 70&#181;m, for hepatocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td>centrifuge for cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clips&#160;</td>
			<td>Teleflex Medical</td>
			<td>REF 523700</td>
			<td>Titanium Clips for spleen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Primovert&#160;</td>
			<td>microscope for cell observation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mtd2 mice</td>
			<td>N/A</td>
			<td></td>
			<td>Gift from Dr. William A Held at roswell Park Cancer Institute in 2002, maintained in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>BD</td>
			<td>REF 305109</td>
			<td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture</td>
			<td>ETHICON</td>
			<td>J303H</td>
			<td>coated VICRYL suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SV40 T Ag antibody</td>
			<td>Abcam</td>
			<td>ab16879</td>
			<td>anti-SV40 T-antigen antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>BD</td>
			<td>REF 309626</td>
			<td>1 mL TB syringe for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>SIGMA</td>
			<td>T 8154</td>
			<td>Trypan blue solution for cell viability test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Reflex</td>
			<td>reflex9, Part. No. 201-1000</td>
			<td>stainless steel wound clips for wound close</td>
		</tr>
	</tbody>
</table>

an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis

간세포암(HCC)의 전형적인 면역 특성을 다루는 임상적으로 관련된 동물 모델의 부재는 근본적인 메커니즘의 해명과 혁신적인 면역 치료 전략의 개발을 크게 저해했습니다. 인간 HCC를 재량화하는 이상적인 동물 모델을 개발하기 위해, 면역 적격 남성 C57BL/6J 마우스는 먼저 간 섬유증을 유도하기 위해 탄소 사염화물 (CCl4)주사를 받은 다음 젊은 남성으로부터 조직학적으로 정상 인간 세포체를 받습니다. SV40 T 항원 (TAg)-형질전환 마우스 (MTD2) 인트라 비장 (ISPL) 접종에 의해. 사춘기에 수용자 남성 마우스에서 생성 된 안 드로 겐 간 특이적 프로모터의 제어 하에 TAg 발현을 시작. 결과적으로, 전송된 간세포는 암세포가 되고 간 섬유증/간경변의 조정에 있는 종양 질량을 형성합니다. 이 새로운 모델은 간 섬유증/간경변의 맥락에서 인간 HCC 개시 및 진행을 모방하고 면역 기능 장애를 포함하여 인간 HCC의 가장 전형적인 특징을 반영합니다.

TAg-형질전환 마우스로부터 분리된 종인성 간세포(도2)를비장 내 주사에 의해 야생형 마우스의 간에서 파종하였다(도3). 이식된 간세포는 종양 특이적 항원 SV40 TAg(도 5)와 함께 정형암 HCC 종양을 성공적으로 안정적으로 성장시키고 간염증 및 섬유증의 설정에서 성장하였다(도1).
<img alt="Figu...

Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

여기서, 간세포암(HCC)의 전형적인 면역 특징을 재기하는 간암의 임상적으로 관련 있는 뮤린 모델의 개발을 설명한다.

간 염증과 섬유증의 설정에서 발생하는 간세포암의 종양 성 간세포 유도 직교 마우스 모델

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

간세포암 질량 모델을 개발하기 위해 단지 모델을 사용하면 사람들이 종양으로 유도된 면역 편성 및 특정 치료 유도, 종양 면역 반응을 해명하는 데 도움이 될 수 있습니다. 간 섬유증 기술로 발전하는 치열토성 종양은 인간 간암의 임상 적 특징을 모방합니다. 이 방법은 간 세포암 면역학 연구에서 유용한 도구를 제공 할 수 있습니다.이 방법의 시각적 데모는 이 프로토콜의 성공적인 실행에 필수적인 내장 주입과 같은 기술적으로 어려운 단계를 설명할 수 있습니다. 다음으로, 수동으로 남성, 6 ~ 8 주 된 C57 블랙 6 마우스 등쪽 업을 억제합니다. 대체 70%에탄올과 베타딘 스크럽을 사용하여 주사 부위를 세 번 청소하십시오.그런 다음 관면 주사로 160 마이크로 리터의 탄소 테트라 클로리드를 마우스에 전달하고 동물을 홈 케이지로 돌려줍니다. 발가락 핀치에 대한 반응의 부족을 확인 한 후, 5 개월 된 simian 바이러스 40 T 항원 형질 성 마우스에서, supine 위치에 마우스를 배치하고 테이프로 사지를 고정. 리나 알바의 길이를 따라 중간 라인 복강경 절개를 만들고, 간을 적절하게 노출시킬 수 있을 만큼 크고, 창자를 왼쪽으로 대체합니다.열등한 베나 카바를 노출하기 위해 간 위로 해부하십시오. 동맥 클램프를 유도하여 선박을 리디게이트합니다. 나비 바늘을 사용하여 포털 정맥에 정맥에 대한 정맥 내 액세스를 얻고 카테터를 수동으로 고정하십시오.각 솔루션에 대해 주사기를 신중하게 전환하여 15밀리리터 15밀리리터, 0.75%의 콜라게나아제 용액 2개, 카테터를 통한 콜라게나아제 없이 2밀리리터 15밀리리터를 분당 약 8.9밀리리터의 솔루션으로 성공적으로 제공합니다. 관류의 끝에서, PBS의 10 15 밀리리터를 포함하는 50 밀리리터 원추형 관으로 종양 질량을 수확합니다. 방금 입증 된 대로 다음 MTD2 마우스에서 종양을 소비합니다.가위를 사용하여 간을 작은 조각으로 자릅니다. 조직을 신선한 PBS의 새로운 용기로 옮기고 조직으로부터 나머지 혈액을 제거한 후 조직 조각을 5밀리리터 원칼 튜브로 이송하여 완전한 RPMI 배지의 5밀리리터를 함유한다. 조각이 5 밀리리터 파이펫의 끝을 통해 쉽게 들어갈 수 때까지 세척 된 간 샘플을 다진다.튜브에 충분한 매체를 추가하여 30 밀리리터의 최종 볼륨에 도달합니다. 5밀리리터 파이펫을 사용하여 조직 조각을 세절화한 후 70마이크로미터 스트레이너를 통해 새로운 50밀리리터 튜브로 용액을 필터링합니다. 완벽한 매체로 여과기를 여러 번 씻으세요.최종 볼륨을 50밀리리터로 조정하여 추가로 완전한 배지를 추가로 조정합니다. 원심분리기를 멈추고 상체를 탈착하기 전에 원심분리를 최대 50배까지 빠르게 회전시합니다. 그런 다음 계산을 위해 PBS의 20 밀리리터에서 펠릿을 다시 중단합니다.MTD2 간세포의 내장 주입을 위해 마우스 당 220 마이크로 리터의 간세포와 각 받는 동물 당 27 게이지 바늘을 장착 한 밀리리터 주사기를로드합니다. 마취, 탄소 테트라 클로라이드 처리 동물에서 발가락 핀치에 대한 반응의 부족을 확인합니다. 척추 근육 바로 아래에서 시작하여 왼쪽 측면에 1센티미터 절개를 하여 등기둥 익스트림에서 13번째 갈비뼈와 평행하게 만들고 무딘 뾰족한 포셉을 사용하여 비장을 외장화합니다.비장 동맥과 정맥 사이에 두 개의 중간 크기의 티타늄 클립으로 비장을 잘라 비장의 열등한 극으로 200 마이크로 리터를 전달합니다. 페디클의 열등한 분기를 하나의 중간 크기의 클립으로 클립하고 처음에 배치된 두 클립 사이에 비장을 잘라냅니다. 종양 세포로 직접 주입된 비장의 열등한 극을 제거하고 3-0 폴리글락틴(910)을 사용하여 내근육층을 닫기 위해 봉합을 중단하였다.그런 다음 멸균 된 강철 상처 클립을 사용하여 외부 피부 층을 닫고 1킬로그램당 5 밀리그램을 피하시 투여합니다. 입증된 바와 같이 T 항원 형질성 마우스로부터 의 절연 후, 간세포와 종양 침투 세포 모두 관찰될 수 있다. 세포는 전형적으로 처리 후에 거의 100% 생존성을 보여줍니다.야생형 탄소 테트라클로리드 처리 동물로 내장 접종 후 이식된 간세포는 종양 특이적 시미안 바이러스 40 T 항원인정형세포암 종양을 성공적으로 안정적으로 성장시키고 안정적으로 성장한다. MTD2 질량에서 간세포의 실행 가능하고 순수한 인구의 준비와 내장 주사의 정확한 관리는 절차의 성공에 중요합니다. 우리 팀은 자궁 내 및 관면 주사를 통해 받는 쥐로 간세포 접종을 테스트했지만, 이러한 다른 방법은 정형 외 간 세포 암을 유도 할 수 없었다.이 모델은 면역 감시및 종양 진행의 초기 및 후기 단계에서 내성을 위한 조사를 가능하게 하는 독특한 플랫폼을 제공합니다. 탄소 테트라클로리드를 사용할 때는 독성이 있으므로 주의하십시오.

an-oncogenic-hepatocyte-induced-orthotopic-mouse-model-hepatocellular

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

췌장암의 형광 동소 이식 마우스 모델

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

연구

암 연구학

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

대장 암 정위 마우스 모델에서의 순환 종양 세포의 분리

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping Orthotopic Homograft (Syngeneic) Murine 기본 KPC 췌 장 Ductal 선 암의 Cytometry로의

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

뮤린 췌장 연성 선암의 초음파 유도 정형 외 이식

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

종양 침투 T 세포 독성의 정형 외 췌장 종양 및 Ex 생체 특성의 생성

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

인간 유방 상피 세포에 있는 종양원성 변환의 시험관 내 평가

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

췌장암의 정형소 절제술 마우스 모델

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

간세포 암에서 종양-기질 상호 작용을 조사하는 3차원 스페로이드 모델

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

간세포 암종의 동소 마우스 모델에서 비침습적 PET/MR 이미징

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...