RRW 从圣地亚哥 C3 教士踏板资助的合作翻译研究赠款获得了这项工作的支持 (#PTC2017)。

根据本议定书进行的所有动物实验必须经有关机构动物护理和使用委员会 (IACUC) 批准。这里描述的所有程序都已被 IACUC UCSD 批准。
1. 采购受赠动物
注: 科索沃保护团-卢克4580细胞系是由 LSL-KrasG12D/+所产生的肿瘤建立的;LSL-Trp53R172H/+;PDX1;LSL-ROSA26卢克/+鼠标 (KPC), 这是一个在 C57BL/6 背景下的个人电脑的基因工程模型。这一细胞系的优势在于它组成性表达了荧光素酶在原位模型中对肿瘤的监测。然而, 其他细胞系也可以使用, 只要它们符合遗传背景的受体老鼠。
<ol><li>预先确定实验组和每组动物数量。</li>
	<li>选择年龄 (6-10 周的老老鼠推荐) 和性别的动物正在使用的假设和细胞线使用。</li>
</ol>2. 培养细胞
<ol><li>使用 Dulbecco 的改良鹰培养基 (DMEM): 营养混合物 F12 (50:50) 含有10% 胎牛血清 (血清) 和1% 青霉素-链霉素抗生素 (完全生长培养基) 的培养基来生长 KPC-卢克4580细胞。</li>
	<li>如果冰冻, 在肿瘤植入前4天解冻细胞。</li>
	<li>在无菌条件下, 将细胞生长在37摄氏度, 5% CO2和95% 湿度。</li>
	<li>胰蛋白酶处理细胞与0.25% 胰蛋白酶5分钟, 在37摄氏度之前, 他们达到 > 80% 融合, 并中和胰蛋白酶通过增加两次完全生长培养基的体积。 注: 这些细胞如果使用在100% 融合或传代超过12次, 则不能有效地在小鼠体内建立肿瘤。因此, 在最佳融合下, 最好在早期的通道中冻结细胞。</li>
	<li>使用 hemocytometer 计数细胞, 并进行肿瘤诱导如果有足够的数字存在 (0.5-1 x 106细胞/鼠标)。如果没有, 则将单元格扩展为一个通道, 直到获得足够的细胞数。</li>
</ol>3. 诱导平方肿瘤
<ol><li>准备所需材料: P1000 和 P100 吸管和小贴士, 1 毫升注射器, 25G 针, 基底膜基质 (法钢公司), DMEM 基底介质, 1.5 毫升离心管。保持所有材料无菌和冷 (4 °c), 直到程序结束。同时保持头发剪和酒精拭子准备动物使用。</li>
	<li>胰蛋白酶处理 (按步骤 2.4) 和计数细胞, 并并用重悬他们在浓度 2-4 x 107细胞/毫升的冷 DMEM。</li>
	<li>添加等量的100% 法钢公司, 使最终细胞悬浮在50% 法钢公司。最后的体积应相当于每只鼠标所需的50µL 细胞悬浮。准备10% 的过剩, 以容纳在注射器和任何吹打错误的死亡空间。</li>
	<li>整除悬浮在550µL 容量在1.5 毫升离心机管子放置在冰。</li>
	<li>使用抑制剂或手动抑制动物 (6 周大的雄性 C57BL/6 小鼠)。 注: 另一种方法是, 在氧气气体中用2.5% 异氟醚进行麻醉, 使用精密蒸发器的流速为2升/分。</li>
	<li>剃掉注射的部位, 最好是在腿上方的侧面, 使用剪刀。使用酒精拭子, 清洗现场注射两次。</li>
	<li>在一个7毫升的冷注射器中绘制250µL 细胞悬浮液, 最终浓度为 1-2 x 101细胞/毫升, 并通过上下移动活塞来去除任何气泡。将25G 针贴在注射器上, 确保填充针内的空间。</li>
	<li>小心地将针插入到靠近侧面的身体上, 在皮肤下方, 确保针头不会刺穿腹腔或四肢的肌组织。</li>
	<li>使针平行于身体, 使皮肤上的皱纹扁平化。慢慢注射50µL 细胞悬浮液。十年代将针放在注射部位, 允许法钢公司固化以防止渗漏。</li>
	<li>慢慢地取出针保持它平行没有任何侧身运动。用酒精拭子轻轻擦拭注射部位, 将鼠标放到其外壳上。 注: 如果鼠标在手术过程中被麻醉, 监视动物直到它恢复其矫正反应。</li>
	<li>用卡尺定期监测肿瘤的生长情况, 直至直径达到5毫米。</li>
</ol>4. 原位肿瘤的诱导
<ol><li>准备所需材料。高压釜的外科工具, 如剪刀, 手术刀, 针驱动, 尖头和扁平尖钳。保持方便的无菌缝合 (可吸收6-0 和不可吸收的 4-0), 酒精拭子, 发刀, 脱毛膏, 眼润滑剂, 棉纱布, 10% 聚维酮碘溶液, 手术窗帘, 加热垫, 丁丙诺啡, 和麻醉药。</li>
	<li>弄死一个捐赠的老鼠 (最好是相同的背景所需的原位模型) 携带一平方 KPC 肿瘤使用缓慢填充 CO2室。使用无菌钳和 microscissors 在原位植入前不久, 在 BSL-2 罩的不育条件下小心地将该平方瘤切除。在肿瘤切除术中, 注意不要在腹腔内穿刺, 立即用2种无菌 PBS 进行冲洗。</li>
	<li>使用无菌手术刀, 将切除的肿瘤切成1毫米3片, 放在含有冷不育 DMEM 基底培养基的培养皿上, 放入冰上贮存直至植入。</li>
	<li>管理接受者鼠标 0.05-1 毫克/千克丁丙诺啡镇痛平方, 30 分钟手术前。</li>
	<li>使用精确的蒸发器 (或其他麻醉药), 麻醉接受 2-3% 异氟醚的氧 (2 升/分) 的受体小鼠。把老鼠放在37摄氏度的加热垫上, 整个过程。在眼睛上涂抹润滑剂以防止脱水。最初, 通过缺乏令人吃惊的反射来测试麻醉深度, 并通过缺乏踏板反射来确认手术平面麻醉的轻微脚趾捏。在整个手术过程中保持麻醉。</li>
	<li>把鼠标放在它的背部, 轻轻地把它放到右边, 这样腹部的左侧就暴露了。用脱毛膏和纱布清洗鼠标的腹部毛发, 确保无游离毛发进入腹后切口。用3循环10% 聚维酮碘然后用酒精湿巾对皮肤进行消毒, 准备左腹部进行手术。</li>
	<li>使用无菌手术刀, 在皮肤上做一个1.5 厘米横向或斜切口, 1 厘米到中线左侧, 胸腔下方, 稍内侧到脾脏。然后, 通过腹部肌组织扩大切口, 对上覆的浅切口进行镜像。</li>
	<li>用扁平的尖钳定位脾脏, 并从腹腔轻轻地将其从腹腔中具体化。用不育的棉花涂抹器收回脾脏, 并找到脾脏底部附着的胰尾。</li>
	<li>使用扁平尖钳, 侧向胰尾。</li>
	<li>用一根细 (6-0) 无菌缝合针的尖端从捐献者的老鼠身上拿起一个细碎的肿瘤片, 并用无菌钳定位。</li>
	<li>同时轻轻地缩回胰的尾部/身体, 将含有肿瘤片段的缝合针插入胰腺的尾部。通过组织缓慢的缝合, 使肿瘤与胰尾保持接触。根据与组织有关的片段的方向, 应用一个或两个附加缝合线。</li>
	<li>将针头完全从组织中取出, 并在检查出血或渗漏的任何迹象时, 将胰/脾外化的时间延长到六十年代。然后, 用钝钳将它们轻轻地放入腹腔内。</li>
	<li>用6-0 可吸收缝线闭合腹部肌组织, 或连续或间断缝合, 并使用3-0 至6-0 不可吸收的间断缝合闭合上覆皮肤。此时停止麻醉。</li>
	<li>允许鼠标在自己的笼子里恢复食物和水的自由使用。把笼子放在暖气垫上, 以利于恢复。在恢复过程中监控生命体征, 如呼吸、灌注等。</li>
	<li>一旦老鼠恢复, 确认有矫正反射的迹象, 然后将笼子送回正常的房屋。</li>
	<li>治疗丁丙诺啡 0.05-0.1 毫克/千克 8-12 小时手术后, 每 8-12 小时后的过程中需要的疼痛迹象。</li>
	<li>用体内生物发光成像系统监测肿瘤生长 (见材料表)。<ol>
<li>在原位植入术后4天开始进行肿瘤生长, 每周进行两次成像。</li>
		<li>在成像当天, 管理 (腹腔注射) 30 毫克/千克 d-荧光素麻醉 (2-3% 异氟醚氧 (2 升/分)) 接收鼠标10分钟之前的成像。</li>
		<li>荧光素酶活性的图像在无任何发射过滤器的情况下, 在具有自动荧光自由加热阶段的发光成像仪中至少有5的曝光率, 同时仍保持对小鼠的麻醉。</li>
	</ol>
</li>
</ol>5. 平方肿瘤的愤怒
<ol><li>准备所需材料: 方波 electroporator、安全脚开关、电极 (针阵列与镊子电极) 和适配器。保持无菌缝线 (不可吸收 4-0), 酒精拭子, 发刀, 脱毛膏, 眼润滑剂, 棉纱布, 丁丙诺啡, 和麻醉剂也附近。</li>
	<li>使用气体灭菌消毒镊子或针阵列电极。不建议使用热处理。使用玻璃珠消毒器消毒的电极之间的动物。</li>
	<li>在手术前, 将丁丙诺啡 0.05-0.1 毫克/千克治疗肿瘤小鼠30分钟。</li>
	<li>按照步骤 4.4-4.5, 成功地诱导小鼠麻醉, 一旦平方肿瘤种植体达到5毫米直径。</li>
	<li>将麻醉鼠标放在其一侧, 以进入侧面的平方肿瘤。使用一根酒精拭子去除头发用剪刀和干净的皮肤。</li>
	<li>对于平方肿瘤, 使用2针阵列电极。用镊子将皮肤直接提升到肿瘤部位, 并通过与身体平行的皮肤插入电极, 确保它们不会穿透腹腔。一旦通过皮肤, 放置电极的方式, 他们支架肿瘤。</li>
	<li>程序 electroporator 提供100µs 脉冲, 频率为1赫兹, 1500 伏/厘米, 总共150个脉冲。使用脚踏板提供脉冲。在十年代将每组10个脉冲分开, 以允许散热并确认电极的正确位置。 注意: 如果没有麻痹剂的使用, 小鼠将会经历肌肉收缩与愤怒, 可能导致电极的位移, 除非手动保护。</li>
	<li>在完成剂量后取出电极, 不应超过 200s. 记录实际交付的电压, 显示在 electroporator 上。允许老鼠按照步骤 4.14-4.16 的愤怒恢复。</li>
</ol>6. 原位肿瘤的愤怒
注意: 原位肿瘤的愤怒涉及在同一只老鼠上进行第二次生存手术, 因此需要在开始之前从当地 IACUC 得到特别的批准。
<ol><li>准备所需材料: 高压釜的外科工具, 如剪刀, 手术刀, 针驱动, 尖钳, 和扁平尖钳。保持以下附近以及: 无菌缝合 (可吸收6-0 和不可吸收的 4-0), 酒精拭子, 发刀, 脱毛奶油, 眼润滑剂, 棉纱布, 10% 聚维酮碘溶液, 手术窗帘, 暖气垫, 方波 electroporator,安全脚开关, 电极及其适配器, 丁丙诺啡和麻醉剂。</li>
	<li>通过体内成像 (见步骤 4.17), 通过注射30毫克/千克的 d-荧光素腹腔, 对小鼠原位肿瘤生长进行评估。 注: 原位肿瘤是理想的愤怒治疗8至10天后植入, 当肿瘤是清晰可见, 仍然限于胰腺, 因为可以看到在荧光素酶发光图像 (见代表性的结果)。</li>
	<li>按照步骤 4.4-4.9, 找到植入的肿瘤。如果肿瘤不易定位, 则使用钝鼻钳将胃和脾脏轻轻地移动以鉴别肿瘤。</li>
	<li>用钝鼻钳将肿瘤具体化, 并与镊子-电极的铂电极紧密地捕获肿瘤。在步骤5.7 中使用方波 electroporator, 在10个由脚踏开关控制的脉冲中, 传送电穿孔脉冲。</li>
	<li>保持肿瘤的外部性, 至少六十年代后的愤怒, 以监测任何出血的迹象。插入肿瘤回到腹腔, 并关闭切口如前所述 (步骤 4.13-4.16) 和监测鼠标, 直到它恢复。</li>
	<li>观察愤怒对肿瘤生长的影响, 使用体内荧光素酶成像4.17 步。</li>
</ol>

<ol>
	<li>Rahib, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+cancer+incidence+and+deaths+to+2030:+the+unexpected+burden+of+thyroid,+liver,+and+pancreas+cancers+in+the+United+States.">Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States.</a> Cancer Res. 74 (11), 2913-2921 (2014).</li><li>Howlader, N., 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=.">.</a> SEER Cancer Statistic Review, 1975-2013. , (1975).</li><li>Clark, C. E., 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=Dynamics+of+the+immune+reaction+to+pancreatic+cancer+from+inception+to+invasion.">Dynamics of the immune reaction to pancreatic cancer from inception to invasion.</a> Cancer Res. 67 (19), 9518-9527 (2007).</li><li>Lee, E. W., Loh, C. T., Kee, S. T. <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+percutaneous+irreversible+electroporation:+ultrasound+and+immunohistological+correlation.">Imaging guided percutaneous irreversible electroporation: ultrasound and immunohistological correlation.</a> Technol Cancer Res Treat. 6 (4), 287-294 (2007).</li><li>Charpentier, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+electroporation+for+the+ablation+of+liver+tumors:+are+we+there+yet?.">Irreversible electroporation for the ablation of liver tumors: are we there yet?.</a> Arch Surg. 147 (11), 1053-1061 (2012).</li><li>Narayanan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+Electroporation.">Irreversible Electroporation.</a> Seminars in Interventional Radiology. 32 (4), 349-355 (2015).</li><li>Martin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+200+locally+advanced+(stage+III)+pancreatic+adenocarcinoma+patients+with+irreversible+electroporation:+safety+and+efficacy.">Treatment of 200 locally advanced (stage III) pancreatic adenocarcinoma patients with irreversible electroporation: safety and efficacy.</a> Ann Surg. 262 (3), 486-494 (2015).</li><li>Belfiore, M. 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=Percutaneous+CT-guided+irreversible+electroporation+followed+by+chemotherapy+as+a+novel+neoadjuvant+protocol+in+locally+advanced+pancreatic+cancer:+Our+preliminary+experience.">Percutaneous CT-guided irreversible electroporation followed by chemotherapy as a novel neoadjuvant protocol in locally advanced pancreatic cancer: Our preliminary experience.</a> Int J Surg. 21 Suppl 1, S34-S39 (2015).</li><li>Belfiore, G., 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=Concurrent+chemotherapy+alone+versus+irreversible+electroporation+followed+by+chemotherapy+on+survival+in+patients+with+locally+advanced+pancreatic+cancer.">Concurrent chemotherapy alone versus irreversible electroporation followed by chemotherapy on survival in patients with locally advanced pancreatic cancer.</a> Med Oncol. 34 (3), 38 (2017).</li><li>Chu, K. F., Dupuy, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+ablation+of+tumours:+biological+mechanisms+and+advances+in+therapy.">Thermal ablation of tumours: biological mechanisms and advances in therapy.</a> Nat Rev Cancer. 14 (3), 199-208 (2014).</li><li>Wissniowski, T. T., 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=Activation+of+tumor-specific+T+lymphocytes+by+radio-frequency+ablation+of+the+VX2+hepatoma+in+rabbits.">Activation of tumor-specific T lymphocytes by radio-frequency ablation of the VX2 hepatoma in rabbits.</a> Cancer Res. 63 (19), 6496-6500 (2003).</li><li>Eros de Bethlenfalva-Hora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+ablation+suppresses+distant+tumour+growth+in+a+novel+rat+model+of+multifocal+hepatocellular+carcinoma.">Radiofrequency ablation suppresses distant tumour growth in a novel rat model of multifocal hepatocellular carcinoma.</a> Clin Sci (Lond). 126 (3), 243-252 (2014).</li><li>Zerbini, 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=Radiofrequency+thermal+ablation+for+hepatocellular+carcinoma+stimulates+autologous+NK-cell+response.">Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.</a> Gastroenterology. 138 (5), 1931-1942 (2010).</li><li>Ali, M. 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=Activation+of+dendritic+cells+by+local+ablation+of+hepatocellular+carcinoma.">Activation of dendritic cells by local ablation of hepatocellular carcinoma.</a> J Hepatol. 43 (5), 817-822 (2005).</li><li>Fietta, A. M., 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=Systemic+inflammatory+response+and+downmodulation+of+peripheral+CD25+Foxp3++T-regulatory+cells+in+patients+undergoing+radiofrequency+thermal+ablation+for+lung+cancer.">Systemic inflammatory response and downmodulation of peripheral CD25+Foxp3+ T-regulatory cells in patients undergoing radiofrequency thermal ablation for lung cancer.</a> Hum Immunol. 70 (7), 477-486 (2009).</li><li>Jiang, C., Davalos, R. V., Bischof, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+basic+to+clinical+studies+of+irreversible+electroporation+therapy.">A review of basic to clinical studies of irreversible electroporation therapy.</a> IEEE Trans Biomed Eng. 62 (1), 4-20 (2015).</li><li>Neal, R. E., 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=Improved+local+and+systemic+anti-tumor+efficacy+for+irreversible+electroporation+in+immunocompetent+versus+immunodeficient+mice.">Improved local and systemic anti-tumor efficacy for irreversible electroporation in immunocompetent versus immunodeficient mice.</a> PLoS One. 8 (5), e64559 (2013).</li><li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer Cell. 7 (5), 469-483 (2005).</li><li>Safran, M., 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=Mouse+reporter+strain+for+noninvasive+bioluminescent+imaging+of+cells+that+have+undergone+Cre-mediated+recombination.">Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination.</a> Mol Imaging. 2 (4), 297-302 (2003).</li><li>Martin, R. C., McFarland, K., Ellis, S., Velanovich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+electroporation+in+locally+advanced+pancreatic+cancer:+potential+improved+overall+survival.">Irreversible electroporation in locally advanced pancreatic cancer: potential improved overall survival.</a> Ann Surg Oncol. 20 Suppl 3, S443-S449 (2013).</li><li>Neal, R. E., Garcia, P. A., Robertson, J. L., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Characterization+and+Numerical+Modeling+of+Tissue+Electrical+Conductivity+during+Pulsed+Electric+Fields+for+Irreversible+Electroporation+Treatment+Planning.">Experimental Characterization and Numerical Modeling of Tissue Electrical Conductivity during Pulsed Electric Fields for Irreversible Electroporation Treatment Planning.</a> IEEE Transactions on Biomedical Engineering. 59 (4), 1076-1085 (2012).</li></ol>

作者没有相关的财务披露。

在这项研究中, 我们已经展示了一个免疫鼠标模型, 可用于研究愤怒对肿瘤生长的影响。目前, 愤怒被用作非热消融技术仅在高度选择的本地高级 PC 患者谁没有远的疾病进展后几个月的术前治疗。因此, 它的使用是有限的, 因为大多数患者与本地先进的 PC 发展遥远的转移疾病20。该模型将作为研究的基础, 以评估愤怒对 PC 使用各种扩展治疗参数和组合的影响。
...

胰腺导管腺癌 (PC) 预计将成为美国癌症死亡的第二个主要原因, 大约 20201。绝大多数被确诊为 PC 的患者最终会死于2的远距离转移疾病。PC 微环境是众所周知的免疫抑制和 chemoresistant。它的硬性基质含有缺乏效应 (抗肿瘤) T 细胞和免疫抑制白细胞的突出, 包括肿瘤相关的巨噬细胞 (TAMs), 髓系衍生抑制单元 (MDSCs) 和调节 T 细胞 (Tregs)3.这些因素的基础是必须制定多式联运战略, 以抵消微环境的这些影响。
愤怒已经被发展成一种非热的肿瘤消融方法。不像热消融技术, 愤怒不会导致快速凝固坏死, 而是导致逐步凋亡细胞死亡4。重要的是胰腺肿瘤, 愤怒是不容易受到 "散热" 的影响, 可以执行右下血管5。这项技术有 510 (k) 清除从 FDA6 , 目前正在临床上使用, 为某些患者局部晚期或边缘切除胰腺癌。在为 PC7发布的最大的愤怒系列中, 被激怒的病人中位数的生存率大约是单纯的以现代化疗治疗的病人的存活率, 而没有切除8,9。
一些研究表明, 热消融诱发了全身免疫反应的其他肿瘤类型 (在楚等。10). 射频消融在动物肿瘤模型中导致 T 细胞浸润11,12, 包括在肝癌患者中激活自然杀伤细胞 (NK) 的增加13, 14, 肺癌患者免疫抑制 Tregs 减少15。更小数量的研究已经检查了免疫, 微环境和伤害反应的愤怒16。愤怒已被证明刺激系统免疫反应的免疫小鼠模型, 其中二次 (对侧) 肾细胞移植的增长减少或预防的愤怒的原发肿瘤两周前17。他们还观察到, 免疫小鼠需要比免疫缺陷小鼠更少的电压进行完全回归。据推测, 愤怒可能导致改进抗原呈现与热消融的凝固性坏死相比, 但这还没有具体研究。
我们已经开发了一个自体鼠标模型的 PC 从 KPC-卢克4580细胞系 (来自北卡罗来纳州大学的 jj 的礼物), 这是从一个在男性 LSL-KrasG12D/+开发的肿瘤;LSL-Trp53R172H/+;PDX1;LSL-ROSA26卢克/+老鼠, 研究愤怒的地方和系统性的影响18,19。这种荧光素表达细胞系免疫, 也癌变在免疫 C57BL/6 小鼠注射的平方或原位, 并可靠地产生肝转移时注入脾脏。我们利用可编程的方波脉冲发生器, 在电压/距离比为1500伏/厘米的情况下, 使用两针阵列探针 (分别为5毫米) 或铂镊子-trodes 到µs 或原位肿瘤, 提供 100-脉冲电流, 在老鼠在小动物中模拟愤怒的影响。

<table><tbody><tr><td>ECM 830 square wave electroporator</td><td>Harvard Apparatus</td><td>BTX # 45-0002 ( 58018-004 )</td><td></td></tr><tr><td>2 needle array electrode</td><td>Harvard Apparatus</td><td>45-0167</td><td></td></tr><tr><td>Safety foot switch</td><td>Harvard Apparatus</td><td>45-0211</td><td></td></tr><tr><td>Platinum Tweezer-trode</td><td>Harvard Apparatus</td><td>&amp;nbsp;45-0486</td><td></td></tr><tr><td>DMEM-F12 media</td><td>ThermoFisher Scientific</td><td>11320-033</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher Scientific</td><td>10437028</td><td></td></tr><tr><td>Trypsin</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354230</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma-Aldrich, Inc.</td><td>792632</td><td></td></tr><tr><td>Lacrilube</td><td>Fisher Scientific&amp;nbsp;</td><td>19090646</td><td></td></tr><tr><td>Buprenorphine</td><td>Fisher Scientific&amp;nbsp;</td><td>NC1292810</td><td></td></tr><tr><td>D-luciferrin</td><td>Perkin Elmer</td><td>122799</td><td></td></tr><tr><td>IVIS&amp;nbsp; Spectrum In Vivo Imaging System</td><td>Perkin Elmer</td><td>124262</td><td></td></tr><tr><td>Mouse strain C57BL/6J</td><td>The Jackson Laboratory&amp;nbsp;</td><td>000664/Black 6</td><td></td></tr><tr><td>Cell line (KPC-Luc 4580)</td><td>J.J. Yeh Lab at University of North Carolina</td><td></td><td></td></tr><tr><td>BD Precisionglide syringe needles</td><td>Sigma-Aldrich, Inc.</td><td>Z192406</td><td></td></tr><tr><td>Alcohol Swab(70% isopropyl alcohol )</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Digital calipers</td><td>ThermoFisher Scientific</td><td>14-648-17</td><td></td></tr><tr><td>Disposable Scalpels, Sterile</td><td>VWR</td><td>21909</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>VWR</td><td>89198</td><td></td></tr><tr><td>Suture Needle, 45 cm, Size 6-0</td><td>Harvard Apparatus</td><td>72-3308</td><td></td></tr><tr><td>Suture Needle, 45 cm, Size 4-0</td><td>Harvard Apparatus</td><td>72-3314</td><td></td></tr><tr><td>Povidone-iodine 10%</td><td>BD</td><td>29900-404</td><td></td></tr><tr><td>Disposable Warming Pad&amp;nbsp;</td><td>KENT SCIENTIFIC CORP.</td><td>TP-3E</td><td></td></tr><tr><td>Mouse Hair Clipper</td><td>KENT SCIENTIFIC CORP.</td><td>CL8787</td><td></td></tr><tr><td>Surgical Drape</td><td>Harvard Apparatus</td><td>59-7421</td><td></td></tr><tr><td>Phosphate-buffered Saline</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr></tbody></table>

a syngeneic pancreatic cancer mouse model to study the effects of irreversible electroporation

胰腺癌 (PC) 是一种在美国每年杀死大约4万名患者的疾病, 它成功地回避了一些治疗方法, 包括有希望的免疫治疗策略。不可逆电穿孔 (愤怒) 是一种非热消融技术, 它诱导肿瘤细胞死亡而不破坏相邻的胶原结构, 从而使该程序在与血管非常接近的肿瘤中进行。与热消融技术不同的是, 愤怒会导致细胞凋亡, 并立即消融导致坏死, 目前在临床上应用于局部高级 PC 的某些患者。在肿瘤微环境中, 像愤怒这样的烧蚀、非靶向的特定程序会引起无数反应。一些研究已经讨论了愤怒对肿瘤生长的影响, 其他肿瘤类型, 但没有人关注 PC。我们开发了一种自体小鼠模型的 PC, 其中皮下 (平方) 和原位肿瘤可以成功地治疗愤怒, 在高度控制的设置, 促进各种纵向研究后程序。这种动物模型作为一个强有力的系统来研究愤怒的影响和方法来提高愤怒的临床疗效。

我们遵循上述程序, 并产生 5-6 周老野生型 C57BL/6 小鼠的平方肿瘤, 接种 1 x 106细胞与50% 法钢公司。当肿瘤大小达到 5-6 毫米直径时, 一些小鼠被安乐死, 他们的肿瘤切除, 并植入原位在接受 C57BL/6 小鼠。愤怒被执行10天后植入, 如图 1中的时间线所示。对残存的小鼠进行了愤怒的实验。
在平方肿瘤, ?...

Watch this Scientific Journal Video about A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation at JoVE.com

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

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

不可逆电穿孔 (愤怒) 是一种非热消融技术, 用于治疗局部晚期胰腺癌。作为一种相对较新的技术, 愤怒对肿瘤生长的影响是不清楚的。我们开发了一种自体老鼠模型, 它有助于研究愤怒对胰腺癌的影响。

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

a-syngeneic-pancreatic-cancer-mouse-model-to-study-effects

Research

JoVE Journal

Cancer Research

11.4K 次观看.  University California San Diego. 不可逆电穿孔 (愤怒) 是一种非热消融技术, 用于治疗局部晚期胰腺癌。作为一种相对较新的技术, 愤怒对肿瘤生长的影响是不清楚的。我们开发了一种自体老鼠模型, 它有助于研究愤怒对胰腺癌的影响。

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

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.

胰腺癌的荧光原位小鼠模型

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 ...

科研

癌症研究

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

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.

毛尿胰腺腺腺腺癌超声引导正射植入

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.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

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

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.

胰腺癌矫形小鼠模型

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

同系小鼠原位同种异体移植物模拟胰腺癌

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

医学

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

原位胰腺癌小鼠模型的动态对比增强磁共振成像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice

Source: Chai, M. G., et al. <a href="https://www.jove.com/v/50395/" target="_blank">Bioluminescent Orthotopic Model of Pancreatic Cancer Progression</a>.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and&#160;in vivo&#160;bioluminescence imaging for non-invasive monitoring of tumor progression. The orthotopic model is suited to both syngeneic and xenograft models and could be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

生物发光原位胰腺癌小鼠模型:一种监测小鼠癌症进展的非侵入性技术

Source: Chai, M. G., et al. Bioluminescent Orthotopic Model of Pancreatic Cancer Progression.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an ...

JoVE Encyclopedia of Experiments

胰腺癌

Assays & techniques

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www.jove.com/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

原位同种移植物肿瘤移植:一种生成胰腺肿瘤小鼠模型的技术

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...