저자는 아브너 췌장암 재단의 지원을 받았습니다.

모든 절차는 뉴 사우스 웨일즈 대학의 동물 관리 및 윤리위원회 (17/109A)에 의해 승인되었습니다. 16-19g의 8-10주 나이 든 암컷 아티믹 Balb/c 누드 마우스가 이 프로토콜에 사용되었습니다. 마우스는 마이크로 이솔레이터 케이지에 보관되었고 시판되는 펠릿 식품및 물 광고 리비툼을공급하였다.
1. 정형외 췌장암 이식
<ol><li>이식을 위해 세포를 준비합니다. 먼저, 절차에 필요한 세포 수를 계산합니다(1 x 106 luciferase 태그된 AsPC-1 세포 및 1 x 106 암 관련 인간 췌장 스텔레이트 세포 [CAhPSC]는 각 동물에 대해 필요합니다).<ol>
<li>가습 된 온도 제어 CO2 인큐베이터에서 이러한 세포를 유지하고 일상적인 마이코 플라즈마 테스트를 수행합니다. AsPC-1 및 CAhPSC에 사용되는 배양 배지는 RPMI 1640(300 mg/L-글루타민, 20% v/v 태아 소 혈청, 1% v/v 페니실린/스트렙토마이신) 및 IMDM(4m L-글루타민, 10% v/v 태아 소 혈청, 1% v/v/v 페니실린/연쇄절제술).</li>
		<li>표준 세포 배양 기술을 사용하여 세포를 세포 현탁액으로 시험시화합니다. 트립신 용액의 두 배에서 각각의 완전한 배양 배지를 사용하여 트립신을 중화시다.</li>
		<li>인산염 완충식식염(PBS)으로 이 세포를 두 번 세척하고 50 μL 세포 현탁액에서 1 x 106 AsPC-1 셀과 1 x 106 CAhPSC를 포함하는 혼합물로 다시 중단한다.</li>
		<li>이 서스펜션을 사용할 때까지 얼음 위에 보관하십시오.</li>
	</ol></li>
	<li>절차에 대한 클래스 II 생물 안전 캐비닛을 준비합니다. 멸균 플라스틱 커튼에 겹쳐진 가열 매트를 사용하십시오. 시술 중 배율의 경우 2.5x에서 3.5배 배율 수술용 루페 쌍을 사용하십시오.</li>
	<li>직경 1cm의 구멍을 거즈 면봉으로 자르면 지갑 끈 면봉을 준비합니다. 지갑 끈 봉합사로 이 구멍을 고정하십시오. 모든 미세 편조 봉합사는 이것에 사용할 수 있습니다 (예를 들어, 5/0 폴리 글리콜산 봉합사). 조임 후 느슨한 매듭이 제자리에 머무를 수 있으므로 편조 봉합사 소재를 추천합니다. 이것은 도 1a에설명되어 있습니다.</li>
	<li>쥐를 80 mg/kg의 케타민과 10 mg/kg의 자일라진 주사로 마취시킵니다.</li>
	<li>투여 5 mg/kg enrofloxacin 항생제 예방, 2.5 mg/kg 플루닉스진 진통 및 1 mL의 0.9% 식염수 피하.</li>
	<li>일단 마취되면, 마우스를 멸균 장에 놓고 포비도네 요오드를 적용한 다음 피부 준비를 위해 70% 에탄올을 적용합니다.</li>
	<li>복부의 왼쪽 두개골 사분면의 피부에 세로 절개를 한 다음 집게 사이의 근육 층을 절개하여 복부를 입력합니다.</li>
	<li>29 G 인슐린 주사기를 50 μL의 세포 서스펜션으로 로드합니다-이것은 인젝트당 1 x 106 CAhPSC 및 1 x 106 루시파라제 태그된 AsPC-1 세포와 동일합니다. 사출 장치에 장착합니다. 이 주입 장치의 설계 및 기능은 그림 1b와 그 전설에 자세히 설명되어 있습니다.</li>
	<li>지갑 끈면봉을 복강경 절개 위에 놓고 비장과 췌장 꼬리를 이 면봉의 개통을 통해 외부화합니다. 지갑 끈을 조여 췌장의 본체를 부드럽게 둘러싸고 췌장 꼬리를 주입합니다. 거즈가 췌장에 대접하는 동시에 수축하지 않을 정도로 단단하게 하는 것이 중요합니다.</li>
	<li>한 쌍의 집게를 사용하여 췌장의 꼬리를 잡고 측면 의 장력을 부드럽게 배치합니다. 얕은 각도에서 바늘로 복부 간음 표면에 구멍을 뚫은 다음 주사 장치와 함께 느리고 제어 된 방식으로 췌장에 세포 현탁액을 주입합니다.</li>
	<li>주입 과정에서, 신중하게 누출에 대 한 관찰-주사 사이트 주위 (역류에서) 췌 장 소의 반대편에 (관통 및 관통의 경우). 눈에 보이는 누출이 발생하면 주사를 멈추고 주사기에 남은 주사의 양을 확인하여 누설의 양을 기록하십시오. 누설이 소량(<10 μL)인 경우 거즈로 누설을 흡수한 다음 바늘을 다른 췌장 로뷸러로 재배치하여 주사를 완료합니다.</li>
	<li>비장과 췌장을 교체하고 복부벽을 5/0 폴리글리콜산 봉합사로 연속하여 닫습니다. 클립으로 피부를 닫습니다.</li>
	<li>마취에서 회복 될 때까지 따뜻하게 된 케이지에서 마우스를 모니터링합니다. 일단 깨어 있고 경고하면 마우스를 다시 케이지로 옮습니다.</li>
</ol>2. 암 절제술 수술 : 실산 췌장 절제술 및 비장 절제술
<ol><li>이식과 관련하여 절제의 타이밍은 실험 프로토콜에 따라 달라질 수 있습니다. 일반적으로, 종양이 절제 전에 적어도 3 주 동안 성장하도록 허용하지만, 특정 이식 된 암 세포주에 대해 경험적으로 최적화.</li>
	<li>절제 수술 전날, 동물에 대한 생물 발광 이미징을 수행하여 국소화된 1차 종양의 존재를 확인합니다. 이 화상 진찰 연구 결과는 단순히 절제에서 명백한 엑스트라 췌장 질병을 가진 마우스를 제외하기 위하여 이용됩니다. 크기나 복사플럭스를 절제 자격을 결정하기 위한 임계값으로 사용해서는 안 됩니다.<ol>
<li>쥐의 무게와 D-루시페린 내과 함께 주입 (150 mg/kg).</li>
		<li>루시페린 운동 곡선의 성능에 의해 각 실험에 대한 루시페린 주입과 관련하여 이미징 단계의 타이밍을 결정한다. 복사 플럭스가 최대의 90% 이상이되는 기간은 생체 발광 이미징에 대한 최적의 시간을 나타냅니다(이 실험에서 18~26분 후 주입).</li>
		<li>마취를 유도하고 이소플루란(각각 4% 및 3%의 산소)을 사용하여 유지하며 생물발광성 이미징 장치(예를 들어, IVIS Lumina II)를 사용하여 이미징을 수행합니다. 자동 노출 및 비닝 설정을 사용합니다(그러나 예상되는 복사 플럭스에 최적화될 수 있음).</li>
	</ol>
</li>
	<li>절차에 대한 클래스 II 생물 안전 캐비닛을 준비합니다. 멸균 플라스틱 커튼에 겹쳐진 가열 매트를 사용하십시오. 해부 중 배율의 경우 2.5x에서 3.5배 배율 수술용 루페 쌍을 사용하십시오.</li>
	<li>쥐를 80 mg/kg의 케타민과 10 mg/kg의 자일라진 주사로 마취시킵니다.</li>
	<li>투여 5 mg/kg enrofloxacin 항생제 예방, 2.5 mg/kg 플루닉스진 진통 및 1 mL의 0.9% 식염수 피하.</li>
	<li>마우스를 멸균 장에 놓아 수핀 위치에 놓고 포비도요오드를 바르고 피부 준비를 위해 70% 에탄올을 적용합니다.</li>
	<li>복부의 왼쪽 두개골 사분면의 피부에 세로 절개를, 바람직하게는 이전 절개 부위를 통해.</li>
	<li>근본적인 근육 복벽에서 피부를 무뚝뚝하게 해부한 다음 Alm 자가 유지 리트랙터를 배치하여 피부 상처를 열어 놓습니다.</li>
	<li>이전 작업의 봉합선의 한쪽에 집게 사이의 근육 층을 절인 다음 절개를 확장하여 이전 봉합사 라인 전체를 축출한다.</li>
	<li>비장과 말단 췌장을 외부화하고 craniis로 철회하십시오. 췌장의 소달 양상에서, 결장은 필름 접착에 의해 부착된 것을 발견할 수 있다. 이 발견 되면 결장의 결장해제를 무뚝뚝하게 해부합니다.</li>
	<li>조심스럽게 한 쌍의 집게 를 췌장과 비장 용기의 몸에 전달하고이 공간을 엽니다. 이렇게 하면 후속 결찰을 위해 췌장 세그먼트가 확보됩니다.</li>
	<li>췌장의 본체를 티타늄 결찰 클립으로 종양으로 옮기고, 배설물을 소터리와 함께 이것으로 변환합니다. 췌장 그루터기를 제어하는 또 다른 방법은 트랜섹션 전에 5/0 폴리글리콜산 봉합사로 연속성을 유지하는 것입니다.</li>
	<li>췌장을 소생시키고 비장의 두개골 장대와 위 사이의 위장 혈관을 소생시킵니다.</li>
	<li>시편을 제거하고 해모를 확인합니다.</li>
	<li>5/0 폴리글리콜산 봉합사로 복벽을 연속하여 닫습니다. 클립으로 피부를 닫습니다.</li>
</ol>3. 수술 후 관리
<ol><li>즉각적인 포스트 마취 기간 (위의 절차 모두에 대해) 마취에서 회복 될 때까지 따뜻하게 된 케이지에서 마우스를 모니터링하십시오. 일단 깨어 있고 경고하면 마우스를 다시 케이지로 옮습니다. 수술 후 기간에 동물에게 통증과 고통의 징후를 모니터링하십시오. 피하 주사에 의해 0.05 mg/kg buprenorphine를 관리하고 밀접하게 12 시간 동안 동물을 관찰.</li>
	<li>그 후, 체중, 음식 섭취 및 활동에 대 한 매일 쥐를 모니터링. 절개 부위를 검사하고 종양 크기에 대한 각지. 수술 후 일곱 번째 날에 피부 클립을 제거합니다.</li>
	<li>인도적 엔드포인트에 도달하면 마우스를 안락사시합니다. 이러한 인도적 종점은 다음과 같습니다 : 체중 >20%, 치료 할 수없는 고민의 특징 (구부러진 자세, 운동 또는 그루밍의 부족 포함) 및 외부 심백에 의해 추정된 1cm3 보다 큰 종양 크기.</li>
</ol>

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A., 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=Fluorescence-guided+surgery+allows+for+more+complete+resection+of+pancreatic+cancer,+resulting+in+longer+disease-free+survival+compared+with+standard+surgery+in+orthotopic+mouse+models.">Fluorescence-guided surgery allows for more complete resection of pancreatic cancer, resulting in longer disease-free survival compared with standard surgery in orthotopic mouse models.</a> Journal of the American College of Surgeons. 215 (1), 126-135 (2012).</li><li>Ni, X., Yang, J., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging-guided+curative+surgical+resection+of+pancreatic+cancer+in+a+xenograft+mouse+model.">Imaging-guided curative surgical resection of pancreatic cancer in a xenograft mouse model.</a> Cancer Letters. 324 (2), 179-185 (2012).</li><li>Hiroshima, Y., 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=Hand-held+high-resolution+fluorescence+imaging+system+for+fluorescence-guided+surgery+of+patient+and+cell-line+pancreatic+tumors+growing+orthotopically+in+nude+mice.">Hand-held high-resolution fluorescence imaging system for fluorescence-guided surgery of patient and cell-line pancreatic tumors growing orthotopically in nude mice.</a> Journal of Surgical Research. 187 (2), 510-517 (2014).</li><li>Hiroshima, Y., 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=Metastatic+recurrence+in+a+pancreatic+cancer+patient+derived+orthotopic+xenograft+(PDOX)+nude+mouse+model+is+inhibited+by+neoadjuvant+chemotherapy+in+combination+with+fluorescence-guided+surgery+with+an+anti-CA+19-9-conjugated+fluorophore.">Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.</a> PLoS One. 9 (12), 114310 (2014).</li><li>Hiroshima, Y., 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=Fluorescence-guided+surgery+in+combination+with+UVC+irradiation+cures+metastatic+human+pancreatic+cancer+in+orthotopic+mouse+models.">Fluorescence-guided surgery in combination with UVC irradiation cures metastatic human pancreatic cancer in orthotopic mouse models.</a> PLoS One. 9 (6), 99977 (2014).</li><li>Metildi, C. A., 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=Ratiometric+activatable+cell-penetrating+peptides+label+pancreatic+cancer,+enabling+fluorescence-guided+surgery,+which+reduces+metastases+and+recurrence+in+orthotopic+mouse+models.">Ratiometric activatable cell-penetrating peptides label pancreatic cancer, enabling fluorescence-guided surgery, which reduces metastases and recurrence in orthotopic mouse models.</a> Annals of Surgical Oncology. 22 (6), 2082-2087 (2015).</li><li>Metildi, C. A., 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=Fluorescence-guided+surgery+with+a+fluorophore-conjugated+antibody+to+carcinoembryonic+antigen+(CEA),+that+highlights+the+tumor,+improves+surgical+resection+and+increases+survival+in+orthotopic+mouse+models+of+human+pancreatic+cancer.">Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer.</a> Annals of Surgical Oncology. 21 (4), 1405-1411 (2014).</li><li>NCCN Guidelines: Pancreatic Adenocarcinoma. National Comprehensive Cancer Network Available from: <a target="_blank" href="https://www.nccn.org/professionals/physician_gls/pdf/pancreatic.pdf">https://www.nccn.org/professionals/physician_gls/pdf/pancreatic.pdf</a> (2019)</li><li>Maithel, S. K., Allen, P. J., Jarnagin, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Blumgart's Surgery of the Liver, Biliary Tract and Pancreas, 2-Volume Set (Sixth Edition). , 1007-1023 (2017).</li><li>Egberts, J. 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=Dexamethasone+reduces+tumor+recurrence+and+metastasis+after+pancreatic+tumor+resection+in+SCID+mice.">Dexamethasone reduces tumor recurrence and metastasis after pancreatic tumor resection in SCID mice.</a> Cancer Biology &amp; Therapy. 7 (7), 1044-1050 (2008).</li><li>Xu, Z. 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=Targeting+the+HGF/c-MET+pathway+in+advanced+pancreatic+cancer:+a+key+element+of+treatment+that+limits+primary+tumor+growth+and+eliminates+metastasis.">Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumor growth and eliminates metastasis.</a> British Journal of Cancer. , (2020).</li><li>Pothula, S. P., 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=Targeting+the+HGF/c-MET+pathway:+stromal+remodelling+in+pancreatic+cancer.">Targeting the HGF/c-MET pathway: stromal remodelling in pancreatic cancer.</a> Oncotarget. 8 (44), 76722-76739 (2017).</li><li>Pothula, S. P., 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=Hepatocyte+growth+factor+inhibition:+a+novel+therapeutic+approach+in+pancreatic+cancer.">Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer.</a> British Journal of Cancer. 114 (3), 269-280 (2016).</li><li>Giri, B., 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=An+Immunocompetent+Model+of+Pancreatic+Cancer+Resection+and+Recurrence.">An Immunocompetent Model of Pancreatic Cancer Resection and Recurrence.</a> Journal of Gastrointestinal Surgery. , (2020).</li><li>Allen, V. B., Gurusamy, K. S., Takwoingi, Y., Kalia, A., Davidson, B. 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저자는이 프로젝트에 대해 공개 할 것이 없습니다.

췌장암의 절제 성 교정 마우스 모델은 보조 및 신아주반트 치료의 검사를 허용하기 때문에 중요합니다. 이것은 수술이 가장 효과적인 처리남아 그러나 재발의 고위험과 연관되는 췌장암에서 특히 중요합니다. 이 논문은 신중하게 절제술로 치료 할 수있는 췌장암을 안정적으로 생성하는 방법을 설명하며, neoadjuvant / 보조 치료가 필요한 임상 시나리오를 복제합니다.
기?...

췌장 덕트 아데노암 (췌장암 [PC])은 가난한 예후1과관련이 있다. 외과 절제술은 PC를 위한 유일한 잠재적으로 치료 처리 남아 있고 초기 단계 질병으로 제출하는 환자를 위해 고려되어야 합니다. 불행히도, R0 절제(즉, 종양이 없는 절제 마진)에서도 재발률(국소 또는 검출되지 않은 전이성 질환)은2,3이다. 따라서, 전신 보조요법은 절제술4를겪는 거의 모든 환자에서 표시된다. 더욱이, 네오아드주반트 요법은 이제 경계선 절제암에대해서만 권장되지만, 그 증상은 많은 임상 연구의 초점이될 정도로 확장되고 있다5,6,7,8. 절제술을 포함하는 PC를 위한 새로운 치료 접근을 개발하기 위하여는, 이 접근은 정확하게 임상 설정을 회수하는 전 임상 모형에서 첫째로 평가될 필요가 있습니다.
PC의 직교 마우스 모델은 과거에 약물 치료9,10을테스트하는 데 자주 사용되어 왔다. 이들 중 많은 마우스 췌장에 혼자 암 세포의 주입에 의해 생산 되었다, PC의 특징인 눈에 띄는 스트로마 부족 종양의 결과. 최근에는 인간 PC와 인간 췌장 스텔레이트 셀(PC내 콜라주기의 1차 생산업체인 PSC)의 혼합물을 주입하여 처음 개발한 것과 같은 공동 주입 직교 모델들이 정기적으로 사용되어11,12로정기적으로 사용된다. 이러한 암 및 기질 세포의 공동 주입에 의해 생성된 종양은 (i) PC의 암 원소 및 특성 기질(desmoplastic) 성분을 모두 나타내며, (ii) 향상된 암세포 증식 및전이(11). 따라서 이 모델은 인간 PC와 매우 유사합니다. 정형PC의 다수의 절제 모델이13,14,15,16으로설명되었지만, 인간에서 췌장 절제술의 임상적 현실을 본 모델만큼 정확하게 반영한 사람은 없으며, 따라서 보조제 또는 신아주반트 치료를 테스트하기 위한 최적이 아니다.
제시된 마우스 모형의 목적은 (i) 성공적으로 자궁경부 췌장암을 이식하는 방법을 보여주기 위한 것이었으며, 부주의한 회대 보급을 최소화하고 (ii) 이후에 암을 완전히 절제술하였다. 이 문서는 이 기술의 팁과 잠재적인 함정을 강조합니다.

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

췌장암(PC)의 수술을 고려중인 환자에서 보조 및/또는 신아주반트 요법을 연구하는 만족스러운 동물 모델의 부족이 있습니다. 이 결핍을 해결하기 위해, 우리는 말단 pancreatectomy 및 비장 절제술 다음에 PC의 직교 이식을 포함하는 마우스 모델을 설명합니다. 이 모델은 보조 및 네오 보조 제 설정에서 다양한 치료 접근법의 연구에 안전하고 적절하게 유연하다는 것이 입증되었습니다.
본 모델에서 췌장종양은 인간 췌장암세포(luciferase-tagged AsPC-1) 및 인간암 관련 췌장 스텔레이트 세포를 Balb/c 아티믹 누드 마우스의 황실 췌장에 이식하여 먼저 생성된다. 3 주 후, 암은 재복복절, 해장 판 절제술 및 비장 절제술에 의해 제복됩니다. 이 모델에서, 생물 발광 화상 진찰은 암 발달의 진행 및 절제술/처리의 효력을 따르기 위하여 이용될 수 있습니다. 절제술 후 보조 요법을 받을 수 있습니다. 대안적으로, 네오아드주반트 치료는 절제 전에 투여될 수 있다.
45마우스의 대표적인 데이터가 제시된다. 모든 마우스는 hemostasis의 문제 없이 성공적인 말단 pancreatectomy/비장 절제술을 겪었습니다. 5mm를 초과하는 거시적 근위 췌장 마진은 43(96%)에서 달성되었습니다. 마우스. 췌장 절제술의 기술적 성공률은 100%였으며 0%의 조기 사망률과 이환율이었습니다. 절제술 후 일주일 동안 동물중 누구도 죽지 않았습니다.
요약하자면, 우리는 임상 시나리오를 모방하는 마우스에 있는 췌장암의 외과 절제술 모형을 위한 강력하고 재현가능한 기술을 기술합니다. 이 모델은 보조 및 신아주반트 치료의 테스트에 유용할 수 있다.

59마리의 연속 마우스가 이식 수술을 받았습니다. 총 누출은 8명(14%)에서 발생했습니다. 마우스. 주사 시의 누설 정도는 프로토콜 섹션에 상술한 바와 같이 추정된다. 이러한 이식된 종양이 증가할 수 있도록 3주 후, 절제 전 생물발광 이미징은 절제 전에 총 전이성 질환을 가진 마우스를 배제하기 위해 수행되었다. 45명 (76%) 마우스는 외과 절제술을 받았습니다.
모든 45 (100%) 마?...

Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

임상 맥락에서, 국소췌암을 가진 환자는 부주 처리를 선행한 pancreatectomy를 겪게 될 것입니다. 여기에서 보고된 이 프로토콜은 말단 판생성 절제술 및 비장 절제술다음에 췌장암의 정형소 이식을 통해 누드 마우스에서 이 임상 시나리오를 모델링하는 안전하고 효과적인 방법을 확립하는 것을 목표로 합니다.

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 ...

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

10.0K 조회수.  University of New South Wales. 임상 맥락에서, 국소췌암을 가진 환자는 부주 처리를 선행한 pancreatectomy를 겪게 될 것입니다. 여기에서 보고된 이 프로토콜은 말단 판생성 절제술 및 비장 절제술다음에 췌장암의 정형소 이식을 통해 누드 마우스에서 이 임상 시나리오를 모델링하는 안전하고 효과적인 방법을 확립하는 것을 목표로 합니다.

비디오: An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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 ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

돌이킬 수 없는 Electroporation의 효과 공부 하는 Syngeneic 췌 장 암 마우스 모델

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

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 ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

생체 내 전파 및 배양 VX2 세포에서 만든 후각 성 림프절증을 가진 정형 외 자궁 내막 암 모델

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

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

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

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 ...

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 ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

초음파 유도 주사에 의한 췌장암 세포의 동종 이종이의 생성

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...