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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

摘要

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

引言

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2.

The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16.

Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

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研究方案

描述的所有研究方案已经由芝加哥机构审查委员会的大学（IRB）审查。知情同意每位患者的术前和研究经芝加哥IRB的大学。生物安全柜型和手套应处理为保护人体组织时，减少污染细胞的危险中。

1.隔离小学未转化的基质细胞与文化

  1. 人体组织收集和准备。
    1. 获得人网膜，2厘米3，腹部手术过程中移除的样本，并立即浸入组织在RT中的磷酸盐缓冲盐水（PBS）（图2A）。一块网膜3厘米×2cm的一般得1万小学人类间皮细胞（HPMC）和20万人类纤维原细胞或正常网膜成纤维细胞（NOF）。
    2. 降速的PBS将组织浸渍于0.5×g离心3分钟尽快后收集（<2小时的PBS），并在50ml锥形管中的组织转移到新鲜的20个ml的PBS中。通过在一个直径为15厘米，无菌培养皿（图2B）手术刀切碎切碎组织成5毫米3块 。
  2. 原代人腹膜间皮细胞分离8
    1. 以分离的HPMC，转移组织糜到50ml锥形管中，并等待1分钟为固体片浮到顶部 （图2C＆D）。 HPMC和红血细胞（RBC）将存在于底部的液体。使用移液管从管的底部，并进入一个新的锥形管中除去液体。旋液向下0.5 XG的3分钟，并吸从HPMC /红细胞沉淀上清。
      1. 重复这个过程三次:加20毫升的PBS到组织糜，让组织上升，从一个吸管，进入HPMC / RBC管底部去除液体，降速，并取出上清。毕竟自旋完成并且将上清液吸出，将丸粒将包含HPMC和RBC。
    2. 板的所有单元从沉淀成75厘米2烧瓶中15充满生长培养基毫升（含10％胎牛血清[FBS]，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。
    3. 执行二次PBS洗分离另外的间皮细胞。
      1. 对于二次PBS洗涤，摇动剩余的固体组织在200rpm，37℃下在20毫升PBS中10分钟，然后等待1分钟为组织浮到顶部。除去从管的底部的液体进新管中，离心，在0.5×g离心3分钟，吸出上清液。
      2. 板上的所有的HPMC / RBC从二级PBS洗涤，并在单独的75cm 2的烧瓶在15毫升完全培养基中。
    4. 要排除任何ř通过体积的PBS溶液:emaining HPMC，摇动组织糜在200rpm，37℃度10分钟在20毫升1:1的0.25％胰蛋白酶的25mM EDTA中。允许组织上升，收集液体在试管的底部到一个新的50ml管中，和自旋向下在0.5 GX 3分钟。
      1. 吸出上清液和板块全部HPMC / RBC在一个单独的75cm 2的烧瓶在15毫升完全培养基中。
    5. 培养在37℃和5％CO 2在加湿环境中铺板的细胞。饲料铺板的细胞中加入15ml充满生长培养基上天3和5不除去用过的培养基。 HPMC可分裂前5-7天培养。
      1. 使用在所有的实验低传代HPMC（最多2代），以尽量减少去分化原来的表型和修改。最好是使用宽视场显微镜用塑料微分干涉对比功能，或相差capabilitie 4-20x物镜20倍的目标秒（在显微镜相衬滤波器）用于取细胞的所有图像，和10x物镜（）在明场来分析免疫组织化学3,3'-二氨基联苯胺（DAB）染色。
    6. 确认HPMC是立方形，并进行免疫组化8（ 图3A，C，E）表达细胞角蛋白8和波形。
  3. NOF隔离8
    1. 制备出10毫升胶原酶III型溶液（10倍）证券的通过组合1:1:100×青霉素 - 链霉素的1:1混合物:胶原酶III型的1500个活性单位/毫升:透明质酸酶在PBS 714单位活性/毫升。
    2. 在10ml的10倍胶原酶III型溶液稀释于无血清培养基（DMEM，用1％的MEM维生素[93毫克/升]摇动剁碎网膜组织后保留的HPMC隔离在200rpm，37℃进行6小时， 1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。消化的组织的外观不透明，并且可以具有一些纤维碎片（图2E）。
    3. 离心溶液含有NOF细胞 在悬浮液中，在0.5×g离心3分钟，在室温，吸出上清液和板在75厘米2烧瓶中的沉淀在13ml充分生长培养基。
    4. 删除旧的媒体24小时后，用15毫升新鲜完全培养基更换。 NOF可以培养分裂前1-3天。注意NOF的扩散将停止，当细胞达到汇合。
    5. 确认原代人成纤维细胞是平的，细长的细胞和表达波形蛋白，但不通过进行免疫组化8角蛋白8（图3B，D，F）。使用低传代诺夫的实验（最好3代之前），以尽量减少去分化 与原始表型的修饰。使用宽视场显微镜用塑料DIC能力20倍的目标采取细胞的所有阶段图像，而在明场一个10倍的目标，分析免疫组化DAB染色。

2.电镀的器官文化8

  1. 从75厘米培养瓶中通过用10ml的PBS，接着毫升0.25％胰蛋白酶3/25毫摩尔EDTA不超过5分钟，漂洗释放NOF。中和胰蛋白酶具有完全生长介质中的至少3倍的体积。
  2. 转移胰蛋白酶的细胞到50ml锥形管中，离心，在0.5 GX 3分钟。除去上清液，并在5毫升完全培养基带来电池备份。使用细胞计数器计数从培养烧瓶中回收细胞。
  3. 稀释细胞在全生长培养基的适当体积到板每100微升2,000-4,000 NOF到黑色，透明底的96孔板。电镀前加入0.5微克/ 100微升鼠尾I型胶原的细胞混合物。让细胞在37℃，坐在5％CO 2在加湿环境中至少4小时，或直至细胞附着在板表面上。
  4. 从利用在步骤2.1和2.2中描述的相同的方法培养瓶中释放的HPMC。稀释细胞在全生长培养基的合适量为板材每50μl10,000-20,000的HPMC在NOF的顶部已经镀有Ⅰ型胶原蛋白在96孔板中。让细胞在37℃，开始实验前坐在5％CO 2在加湿环境至少18小时。
  5. 培养绿色荧光蛋白（GFP）在整个范围内生长培养基-expressing HeyA8卵巢癌细胞。 HeyA8卵巢癌细胞通过表达桡足类cGFP（PCDH-CMV-MCS-EF1）的慢病毒载体表达的荧光标记。 HeyA8卵巢癌细胞应该是在使用的当天（对数生长期）80-90％汇合。从培养板释放细胞和利用在步骤2.1-2.2为原代细胞所描述的相同的方法计数。
  6. 允许卵巢癌细胞完全生长培养基以回收15-20分钟，在37℃下在一锥形管与盖子松松地密封。

3，附着力测定8

  1. 恢复后，通过在0.5×g离心纺丝3分钟沉淀卵巢癌细胞，然后除去上清液和稀释细胞在无血清培养基的适当体积达到50000个细胞/100μl的的浓度。
  2. 倒置96孔板用HPMC / NOF器官型培养以除去用过的培养基，并在无血清培养基中添加100微升癌细胞悬浮液的每个孔中。孵育板在37℃在5％CO 2在加湿环境进行30分钟至4小时。
  3. 倒置96孔板以除去介质和非粘附细胞。使用多管移液器在低功耗仔细，轻轻吸管100微升PBS到每口井，并再次翻转板。重复PBS洗涤一次。
  4. 倒置以除去PBS中，并加入100微升4％多聚甲醛至每个孔中。允许板坐20英里n至固定细胞。 20分钟后，反转板，并添加100微升PBS中。
  5. 需要注意的是井的总荧光可以报告或细胞的数量进行量化。对于定量，一式两份使用HeyA8细胞以1百万个细胞/ ml的浓度的第一板的标准曲线。
    1. 通过停转1百万个细胞，除去培养基，并允许他们坐在在1ml的4％多聚甲醛20分钟使标准曲线。
    2. 自旋细胞再次调出1毫升的PBS（重新计票的细胞，以确认100万/ ml浓度）。板50000，40000，30000，20000，万，5000，1000，和0细胞到每个孔中一式两份，并加入PBS中的50微升每孔的最终总体积。
  6. 底部读取荧光酶标仪（激发= 488纳米，发射= 528纳米）的培养板，比例高的井（标准曲线上50,000）。使用标准曲线计算有多少卵巢癌细胞粘附于organotyp在每个孔中（图4A-C）的集成电路培养。

4.增殖试验10

  1. 后的卵巢癌细胞恢复（参见步骤2.5和2.6以上），并在0.5×g离心纺丝3分钟沉淀细胞，然后稀释所述细胞中的1％FBS的培养基的适当体积（DMEM有1％FBS，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）来实现4000个细胞的浓度/ 150微升。
  2. 用HPMC / NOF器官型培养倒置96孔板以除去用过的培养基，并在1％的添加150微升癌细胞悬浮液的FBS的介质到每个孔中。孵育板在37℃在5％CO 2在72小时的潮湿环境。
  3. 底上读取天（激发= 488纳米，发射= 528纳米）的荧光读板器一旦培养板，评估相对增殖的细胞。继续测定96小时后，改变培养基在48小时（图5A-C）。要小心，不要改变介质时（反相板为首选），扰乱文化。

5.侵袭试验8

  1. 电镀前的3D培养，在200μlPBS中添加7.5微克大鼠尾胶原I到一个24孔培养板插入件（8微米孔径）。让平板孵蛋O / N，然后小心翼翼地用吸管直接电镀HPMC / NOF器官文化到胶原蛋白涂层刀片（步骤2，上文）之前删除的PBS。
  2. 制备卵巢癌细胞如在步骤3.1。到板的卵巢癌细胞上的刀片的器官型培养，小心使用吸管从刀片的顶部除去过量的介质。在无血清培养基中加入100μl卵巢癌细胞悬浮液的每个孔中。
  3. 添加750微升全生长培养基到远低于插入物以诱导癌细胞vasion到器官文化。孵育板在37℃在5％CO 2在24小时的潮湿环境。
    注意:卵巢癌细胞侵入器官培养将跨越所述插入并保持在插入件的底侧。
  4. 24小时后，用抓钳插入并简要冲洗在PBS的烧杯中，然后将插入到一个空的好。擦洗每个插入用棉签的两侧的顶部为总共1分钟。迅速将插入到含有每750微升4％多聚甲醛以及一盘。允许每个插入到传送插入到孔中填充有750微升的PBS之前坐在低聚甲醛10分钟。
  5. 查看侵入细胞（粘附并固定到插入件的底部），用显微镜在2.5倍的放大倍率。当整个井是在视野内，调整放大倍数10倍，并采取5张图片，一个靠近中心和1中的井的每个象限，避免采取这一过于接近的边缘图像。按照同样的模式为每口井。
  6. 使用制造商的方案来计算在每个图像单元的数量根据其协议来获得细胞每场，每孔（图6A-C）的侵入的平均数。

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结果

器官型培养物组装通过首先混合主人成纤维细胞与I型胶原蛋白，然后与间皮细胞的5倍的数量覆盖该培养。将培养物温育至少18小时，加入前卵巢癌细胞，研究粘附，浸润或增殖。每个测定重复与多个（N = 3-5），从不同的患者和许多井获得3D培养在每种条件下进​​行了测试的粘合（N = 5），增殖（N = 5）和侵入测定（n = 3时） 。卵巢癌细胞附着在三维器官型培养4小时（图4）

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讨论

成立腹膜微环境的一个器官模型来评估两者的细胞和先天在卵巢癌传播微环境的组件的单独和集体功能（多个）。提供了用于镀敷和自定义三维器官型培养来研究卵巢癌的细胞粘附，增殖和侵袭的具体协议。原代人网膜间皮细胞和成纤维细胞从患者分离并使用在早期通道保持正常形态和患者的变化。在这种模式下，正常成纤维细胞网膜和ECM（通常胶原I型）的分层与伯人腹膜间皮细胞单层。该模型...

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披露声明

The authors have nothing to disclose.

致谢

We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

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材料

NameCompanyCatalog NumberComments
1. Isolation and culture of primary cells
PBSFisher ScientificSH3001304
Single-edged razor bladesFisher Scientific12-640
15 cm culture dishesBD Biosciences353025
Glass flask??
Fetal Bovine Serum (FBS)Life Technologies16000044_3616914956
DMEM with L-GlutamineCorning10-013-CV
MEM VitaminsCorning25-020-Cl
MEM Nonessential amino acidsCorning25-025-CI
Penicillin-StreptomycinCorning30-002-CI
Shaker Thermo-FisherMaxQ 4450
CentrifugeEppendorf5702
IncubatorThermo-FisherForma Series II Water Jacketed CO2 Incubator Model 3100
Trypsin EDTA, 1x (0.25%)Corning25-053-CI
HyaluronidaseWorthington BiochemicalLS002592
T-75 FlasksBD Biosciences353136
T-175 FlasksBD Biosciences353112
Pipet tipsRaininP2, P10, P20, P200 and P1000
Pipet tipsCorningFiltered tips P2, P10, P20, P200 and P1000
Name of Reagent/ EquipmentCompanyCatalog NumberComments/Description
2. Plating 3D culture
Cell CounterInvitrogenCountess
Countess Cell Counting Chamber SlidesInvitrogenC10313
Trypan Blue Stain (0.4%)Gibco15250-061
Collagen Type I (Rat Tail)BD Biosciences354236
96 well plate, clear bottom, blackBD Biosciences353219
Name of Reagent/ EquipmentCompanyCatalog NumberComments/Description
3. Adhesion assay
Multichannel pipetEppendorfXplorer 300
Paraformaldehyde solution 4% in PBSSanta Cruz Biotechnologysc-281692
Plate readerMolecular DevicesMinimax
Name of Reagent/ EquipmentCompanyCatalog NumberComments/Description
5. Invasion assay
Cell Culture Inserts (8um, 24-well)BD Biosciences353097
Cotton swabsQ-tipscotton swabs
MicroscopeZeissAxiovert 200m
Cell Profilerpublic domain
24 well plateBD Biosciences353047
Name of Reagent/ EquipmentCompanyCatalog NumberComments/Description
6. Antibodies
Anti-Integrin αVβ3 Antibody, clone LM609EMD MilliporeMAB1976
Beta 1 OncosynergyOS2966
Alpha 5 [CD49e]ID Pharmingen555615
Beta 4 [CD104]EMD MilliporeMAB 2058

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