腹膜内卵巢癌转移的定量
Summary
Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.
Abstract
Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.
Introduction
卵巢上皮癌（EOC）是的妇科恶性肿瘤死亡的最常见原因，在美国，2015年估计21290新诊断病例，估计14,180人死亡1。妇女的绝大多数（> 75％）被诊断出患有晚期疾病（III期或IV）特征的弥漫性腹腔转移，预后差。在下面的一线化疗腹腔疾病复发也很常见，并且表示死亡率2,3的一大原因。 EOC发生转移通过同时涉及从原发肿瘤到邻近腹膜器官以及通过解离直接延伸或从原发肿瘤表面为单细胞或多细胞聚集体的细胞的脱落的唯一机制。细胞脱落进入腹膜腔，其中它们抵抗脱离诱导的凋亡4。腹腔腹水的积累是常见的，如脱落的肿瘤细胞阻塞腹膜淋巴引流和Tumors产生改变血管通透性的生长因子。棚肿瘤细胞的一部分附着于腹腔器官和结构，包括肠，肝，大网膜和肠系膜的表面，于是他们锚和增殖，产生多种广泛传播继发性病变3,5。血行转移少见。因此，临床管理通常由细胞减灭术，包括“最佳减瘤”，其定义是切除所有可见肿瘤的（不管多么小）。完整细胞减少与在整体存活6,7-一个显著增加相关，并且与识别和切除病变<0.5厘米挑战相关联。
小动物模型在改善疾病进展的了解，以及在预后生物标志物和新的化疗或联合治疗方法的检测鉴定证明在卵巢癌的研究工具。作为主要的卵巢癌的发病率和转移部位是腹腔，EOC转移的原位模型涉及到腹腔内疾病的分析和表征。虽然已经有在能力图像的肿瘤细胞的最新改进，甚至在单细胞水平，也还存在着在量化EOC的转移性肿瘤负荷显著困难。这些挑战出现因数量，大小和转移灶的解剖位置。此外，存在一个需要标签癌细胞从正常的宿主细胞区分开来。以前的研究已经利用基于抗体的标签协议或肿瘤细胞与荧光素酶8,9的转染。癌细胞的直接荧光标记首次由千岛和同事在1997年10日报道。荧光标记并不需要加入外源基片，并提供精致的肿瘤细胞的特异性，提供了更有效的手段来追踪癌症转移11,12 </suP>。
这里我们描述了使用由红色荧光蛋白（RFP）一同基因原位异种移植模型的转移性疾病的定量分析的光学成像方法-tagged鼠ID8卵巢癌细胞13和免疫能力的C57 / BL6小鼠。我们证明相对肿瘤负担定量的新方法在体内和体外成像结合切除组织自发荧光的。这种方法有在设计用于评估的特定的遗传，后生或微环境改变和/或治疗方法对卵巢癌的器官特异性转移的效果的研究潜在效用。
Protocol
Representative Results
Discussion
相反，使用必须在免疫缺陷小鼠中进行人卵巢癌细胞的研究，上述的协议利用免疫C57 / BL6小鼠和同基因鼠卵巢癌细胞。而这使得在肿瘤进展和转移的免疫浸润的潜在作用的评估，黑头发的腹部表面上的存在使得成像不太敏感。脱毛的使用以除去成像之前毛发增强图像采集，但费时，特别是用于需要纵向成像实验。无毛'裸“鼠可以在这个协议中使用并且不要求基于脱毛-脱毛。此外，裸鼠缺?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).
Materials
| Dulbecco's Modified Eagle Medium | Corning | 10-014-CM | |
| Fetal bovine serum | Gibco | 10437-028 | |
| penicillin/streptomycin | |||
| Insulin-transferrin-sodium selenite media supplement | Sigma | I-1884 | |
| Bruker Xtreme small animal imaging system | Bruker Corp. | ||
| Bruker Multispectral software | Bruker Corp | ||
| lentiviral particles with Red fluorescent protein | GenTarget, Inc. | LVP023 | |
| trypsin for cell culture | Corning | 25-053-CI | |
| PBS | Corning | 21-040-CM | |
| depilatory cream (such as Nair Hair Remover Lotion) | purchases from drugstore | n/a | |
| ImageJ software | http://imagej.nih.gov/ij/ | free download | |
| dissecting tools (forceps) | Roboz Surgical Instrument | RS 5130 | |
| dissecting tools (Scissors) | Roboz Surgical Instrument | RS 5910 |
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