肿瘤细胞外侧尾静脉注射后肺转移的病理分析
Summary
静脉注射癌细胞常用于转移研究，但转移性肿瘤负担可能难以分析。在本文中，我们展示了转移的尾静脉注射模型，并包括一种分析所得转移性肺肿瘤负担的新方法。
Abstract
转移是大多数癌症患者发病和死亡的主要原因，在小鼠中进行临床前建模可能具有挑战性。很少有自发性转移模型可用。因此，涉及尾静脉注射合适细胞系的实验转移模型是转移研究的主要内容。当癌细胞被注射到外侧尾静脉时，肺是它们首选的定植部位。该技术的一个潜在局限性是转移性肺肿瘤负担的准确定量。虽然一些研究人员对预定义大小的大转移酶进行计数和/或包括组织切片后的微转移酶，但其他研究人员则确定相对于正常组织区域的转移性病变区域。当转移负担很高时，这两种定量方法都可能非常困难。在本文中，我们展示了肺转移的静脉注射模型，然后使用图像分析软件量化转移性肿瘤负荷的先进方法。该过程允许研究多个终点参数，包括平均转移大小，转移总数和总转移面积，以提供全面的分析。此外，这种方法已经过美国兽医病理学家学会（SEK）认证的兽医病理学家委员会的审查，以确保准确性。
Introduction
尽管转移是一个高度复杂且低效的过程1，但转移是癌症患者发病率和死亡率的重要因素2。事实上，大多数癌症相关死亡归因于疾病的转移性传播3，4。为了使肿瘤细胞成功转移，它们必须从原发部位脱离，通过相邻的基质侵入，侵入血液循环或淋巴管，行进到次级部位的毛细血管床，外渗到次级组织，并增殖或生长形成转移性病变5。小鼠模型的使用对于进一步了解负责转移性播种和生长的分子机制至关重要6，7。在本文中，我们专注于乳腺癌转移，为此经常使用转基因小鼠模型和移植方法 – 每种方法都有自己的一套优点和局限性。
基因工程乳腺肿瘤模型利用乳腺特异性启动子，包括 MMTV-LTR （小鼠乳腺肿瘤病毒长末端重复）和 WAP （乳清酸性蛋白），以驱动乳腺上皮中转基因的表达8。包括中位T抗原（PyMT）、 ErbB2/Neu、 c-Myc、 Wnt-1和猿猴病毒40（SV40）在内的癌基因已经以这种方式表达9，10，11，12，13，虽然这些遗传模型有助于研究原发性肿瘤的发生和进展，但很少有易转移至远处器官的遗传基因。此外，这些遗传小鼠模型通常比自发或实验转移模型花费更多的时间和成本。鉴于大多数基因工程乳腺肿瘤模型在研究转移方面的局限性，移植技术已成为研究这一复杂过程的有吸引力的方法。这包括原位、尾静脉、心内和颅内注射合适的细胞系。
虽然几种乳腺癌细胞系在将原位注射到乳腺脂肪垫14，15后容易转移，但转移性肿瘤负荷的一致性和可重复性可能是一个挑战，并且此类研究的持续时间可能长达数月。特别是对于评估肺转移，静脉注射到尾静脉通常是一种更具可重复性和时间有效的方法，转移性扩散通常发生在几周内。然而，由于静脉注射模型绕过了转移性级联反应的初始步骤，因此在解释这些研究的结果时必须小心。在这个演示中，我们展示了乳房肿瘤细胞的尾静脉注射以及准确而全面的分析方法。
尽管研究界在了解乳腺癌转移的复杂过程方面取得了重大进展，但据估计，目前有超过150，000名女性患有转移性乳腺癌16。在IV期乳腺癌患者中，>36%的患者有肺转移17;然而，位点特异性模式和转移发生率可能因分子亚型而异18，19，20，21。乳腺癌相关肺转移患者的中位生存期仅为 21 个月，这凸显了确定该疾病的有效治疗方法和新型生物标志物的必要性17。实验转移模型的使用，包括静脉注射肿瘤细胞，将继续推进我们对这一重要临床挑战的了解。当与本方案中描述的数字成像病理学和转移性肺肿瘤负荷分析方法相结合时，尾静脉注射是乳腺癌转移研究的宝贵工具。
Protocol
动物使用遵循俄勒冈州立大学机构动物护理和使用委员会（IACUC）批准的协议2007A0120-R4（PI：Gina Sizemore博士）下的大学实验动物资源（ULAR）法规。 1. 乳腺癌细胞尾静脉注射 制备注射用细胞和注射器 根据要使用的小鼠数量和细胞浓度，接种适当数量的细胞。注意：注射的细胞数量和转移发生的时间将取决于所使用的细胞系，需要优化。在该演示中，将1 x 10…
Representative Results
如果使用未标记的细胞进行尾静脉注射，则可能难以确认肺定植，直到（1）如果可以观察到大转移酶，则尸检时间或（2）如果存在微观转移，则在组织学分析之后。随着广泛的转移性肺肿瘤负担，小鼠将有劳动呼吸。与任何肿瘤研究一样，在整个研究期间应仔细监测小鼠。使用标记细胞是确认尾静脉注射成功的简单方法;因此，在演示中使用了荧光素酶标记的MDA-MB-231细胞。然而，根据实验设计和…
Discussion
随着研究人员继续使用静脉注射肿瘤细胞作为转移的实验模型，缺乏分析由此产生的转移性肿瘤负担的标准实践。在某些情况下，可以在宏观上观察到操作特定细胞系和/或使用化合物时转移性肿瘤负担的显着差异。然而，在其他情况下，如果没有彻底的病理分析，转移性播种和生长的细微差异可能会被忽视或误解。本研究通过包括转移性肺肿瘤负荷分析的综合方法，推进了先前发表的尾静脉注射…
Disclosures
The authors have nothing to disclose.
Acknowledgements
代表性数据由美国国家癌症研究所（K22CA218549至S.T.S）资助。除了协助开发本文报道的综合分析方法外，我们还感谢俄亥俄州立大学综合癌症中心比较病理学和小鼠表型共享资源（主任 – Krista La Perle，DVM，PhD）的组织学和免疫组织化学服务以及病理学成像核心的算法开发和分析。
Materials
| alcohol prep pads | Fisher Scientific | 22-363-750 | for cleaning tail prior to injection |
| dissection scissors | Fisher Scientific | 08-951-5 | for mouse dissection and lung tissue inflation |
| DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate | Fisher Scientific | MT10013CV | cell culture media base for MDA-MB-231 and MVT1 cell lines |
| Dulbecco's Phosphate-Buffered Salt Solution 1x | Fisher Scientific | MT21030CV | used for resuspending tumor cells for injection |
| ethanol (70 % solution) | OSU | used to minimize mouse's fur during dissection; use caution – flammable | |
| Evan's blue dye | Millipore Sigma | E2129 | used at 1 % in sterile PBS for practice with tail-vein injection method; use caution – dangerous reagent |
| Fetal Bovine Serum | Millipore Sigma | F4135 | cell culture media additive; used at 10% in DMEM |
| forceps | Fisher Scientific | 10-270 | for dissection and lung tissue inflation |
| FVB/NJ mice | The Jackson Laboratory | 001800 | syngeneic mouse strain for MVT1 cells |
| hemacytometer (Bright-Line) | Millipore Sigma | Z359629 | for use in cell culture to obtain cell counts |
| insulin syringe (28 G) | Fisher Scientific | 14-829-1B | for tail-vein injections (BD 329424) |
| MDA-MB-231 cells | ATCC | human breast cancer cell line | |
| MVT1 cells | mouse mammary tumor cells | ||
| needles (26 G) | Fisher Scientific | 14-826-15 | used to inflate the mouse's lungs |
| neutral buffered formalin (10%) | Fisher Scientific | 245685 | used as a tissue fixative and to inflate lung tissue; use caution – dangerous reagent |
| NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice | The Jackson Laboratory | 005557 | maintained by OSUCCC Target Validation Shared Resource |
| Penicillin Streptomycin 100x | ThermoFisher | 15140163 | cell culture media additive |
| sterile gauze | Fisher Scientific | NC9379092 | for applying pressue to mouse's tail if bleeding occurs |
| syringe (5 mL) | Fisher Scientific | 14-955-458 | used to inflate mouse lung tissue |
| tail-vein restrainer | Braintree Scientific, Inc. | TV-150 STD | used to restrain mouse for tail-vein injections |
| Trypan blue (0.4 %) | ThermoFisher | 15250061 | used in cell culture to assess viability |
| Trypsin-EDTA 0.25 % | ThermoFisher | 25200-114 | used in cell culture to detach tumor cells from plate |
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Cite This Article
Thies, K. A., Steck, S., Knoblaugh, S. E., Sizemore, S. T. Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells. J. Vis. Exp. (159), e61270, doi:10.3791/61270 (2020).