聚丙烯酰胺凝胶侵袭伪足和牵引力化验的肿瘤细胞
Summary
在肿瘤微机械刚性起着通过增加侵袭伪足活性和肌动球蛋白收缩驱动恶性行为了至关重要的作用。用聚丙烯酰胺凝胶（部分分配），侵袭伪足和牵引力测定可以被用于研究癌细胞的侵入性和收缩特性响应于矩阵刚性。
Abstract
刚性肿瘤组织中一直强烈牵连在调节癌细胞的迁移和侵袭。通过交联组织侵袭性迁移是由肌动蛋白富突起称为侵袭伪足该蛋白水解降解细胞外基质（ECM）提供便利。侵袭伪足的活动已被证明是依赖于ECM的刚性和癌细胞收缩力表明刚性信号可以通过肌动球蛋白收缩调节这些亚细胞结构。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。癌细胞的侵袭性和收缩性能可在体外使用基于不同的刚性聚丙烯酰胺凝胶（分配数量）侵袭伪足和牵引力试验相关。而在两个测定之间一些变型存在，这里介绍的协议提供了用于第创建部分分配的方法在可以在两个测定中使用，并且很容易适应用户的特定生物和技术的需求。
Introduction
肿瘤相关的ECM的刚性已被确定为在通过增加肌动球蛋白收缩1-3找到恶性行为一显著因素。虽然这种效应已主要被证明与乳腺癌细胞，基质刚性已经发现改变从多种癌症4-8表明肿瘤刚度可以以其它类型的癌症中发挥作用的来源的细胞侵入性。渗透侵入迁移过程中，交联组织，癌细胞利用肌动蛋白丰富的被称为侵袭伪足的本地化蛋白酶来灶性降低ECM 9粘合突起。侵袭伪足被认为是侵入细胞的标志，并有牵连的肿瘤细胞浸润和转移10,11。以前的工作已经表明，基质刚度可以调节侵袭伪足数目和通过肌球蛋白II活性和机械敏感蛋白12相关的ECM降解4,12。鉴于肿瘤密度和癌症侵袭性13,14之间的相关性，这些结果表明通过该癌细胞可能响应刚性肿瘤组织通过肌动球蛋白收缩驱动侵袭和转移的机制。
在体外 ECM刚性和体内组织密度已显示调节癌细胞1,15-17侵袭行为。而肌动球蛋白收缩似乎是重要的在此过程中，目前的研究冲突，以转移能力是否相关，增加或减少收缩力6,18-20。此外，目前还不清楚是否这些力量直接介导侵袭伪足的活动21。我们最近发现，肿瘤细胞收缩力是依赖于矩阵的刚度和被预测ECM降解受侵袭伪足5。这些结果表明，蜂窝力可能通过介导侵袭伪足的交流在癌症发展中起重要作用tivity响应于肿瘤微环境的机械性能。
为了关联癌细胞5侵袭和收缩性能，我们修改了协议，用于创建分配数量与以前用于调查的刚性依赖的侵袭伪足的活动4,12,22不同的僵化。通过化学整个部分分配交联的人血浆纤连蛋白，这些修改后的水凝胶可以用作基础为侵袭伪足和牵引力试验，以确保细胞经历了同样的刚性在两个实验5。在侵袭伪足测定中，纤连蛋白提供了天然的结合域为明胶以重叠的ECM链接到部分分配给检测基质降解。在牵引力测定中，纤连蛋白提供的配体的直接的细胞粘连，以检测用来计算蜂窝牵引力微球的位移。这种方法会导致我们所谓的软，硬，和僵化的分配数量绑定到玻璃底菜，并有弹性模量，E，1023，7307和22692帕5的跨度报道了正常和癌变组织23机械性能的范围内。
Protocol
1.准备盖玻片的分配数量清洁12毫米盖玻片低棉绒抹布。 火焰12毫米盖玻片和14毫米盖玻片在35个毫米的玻璃底菜的微孔，通过使用镊子本生灯火焰传递它们。 对待微孔用200μl的0.1N NaOH中进行5分钟，在室温下进行。 吸液和空气干燥的微孔30分钟。 对待微孔用50-100微升3-氨基丙基三甲为10分钟，在室温下于通风柜。这种化学反应，用胶;因此，使用玻璃吸管，不填微?…
Representative Results
在侵袭伪足测定中，侵袭伪足通常标识标记像肌动蛋白和皮层蛋白在细胞体中的点状结构（ 图1）的共定位。既积极降解和非恶化侵袭伪足可以计数和由这些结构是否共定位与缺乏的FITC标记的纤连蛋白（ 图1）的荧光信号的黑色区域是有区别的。侵袭伪足手动计数，而每个单元ECM降解由细胞的轮廓内手动阈值处理这些黑色区域来确定。 在牵引力测定?…
Discussion
我们提出的用于制造部分分配，可以用来作为基础侵袭伪足和牵引力测定法来关联侵入性和收缩的细胞行为的方法。虽然部分分配额早已看惯了对细胞的刚性作用，并计算牵引力18,24,27，该协议是第一个开发基于分配数量与同一刚性平行检测，以响应矩阵相关侵入性和收缩的细胞行为机械性能。正确激活的玻璃底菜盖玻片确保分配数量将绑定到他们，不会脱落。虽然我们和其他人已经依靠…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者什么都没有透露。
Materials
| 3-Aminopropyltrimethoxysilane | Sigma-Aldrich | 281778 | |
| Acrylamide (40%) | Bio-Rad | 161-0140 | |
| Acrylic acid NHS ester | Sigma-Aldrich | A8060 | prepare fresh in fume hood 10 mg/ml in DMSO |
| Alexa Fluor 546 phalloidin | Life Technologies | A22283 | can also use rhodamine |
| Ammonium persulfate | Bio-Rad | 161-0700 | prepare fresh 10% solution in 1X PBS |
| Aqua Poly/Mount | Polysciences | 18606 | use six drops to fill microwells |
| BIS (2%) | Bio-Rad | 161-0142 | |
| Bovine serum albumin | RPI | A30075 | make 3% for blocking solution in 1X PBS and store in 4 °C |
| Coverslips (12 mm) | Fisher Scientific | 12-545-80 | |
| dialysis tubing | Sigma-Aldrich | D9777 | pre-equilibrate in borate buffer for 15-30 min |
| DMEM | Cellgro | 10-013-CV | use to make invadopodia medium |
| DMSO | Sigma-Aldrich | D8418 | use to make acrylic acid NHS ester solution |
| Epidermal growth factor | Life Technologies | PHG0311 | use to make invadopodia medium |
| Ethanol | PHARMCO-AAPER | E200 | dilute with ultrapure water to 70% |
| FBS | Thermo Scientific | SH30070.03 | use to make invadopodia medium |
| FITC | Sigma-Aldrich | F7250 | protect from light |
| Gelatin | Polysciences | 00639 | typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 °C (preheat PBS to dissolve gelatin easily) |
| Glass bottom dishes (35 mm coverslips) | MatTek | P35G-0-14-C | coverslips are uncoated |
| Glutaraldehyde (25%) | Polysciences | 01909 | dilute with 1X PBS to 0.5% |
| goat anti-mouse Alexa Fluor 633 antibody | Life Technologies | A21050 | |
| Human plasma fibronectin | Life Technologies | 33016-015 | add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles |
| KH2PO4 | EMD Millipore | PX-1565-1 | use to make 10X PBS stock |
| mouse anti-cortactin 4F11 antibody | EMD Millipore | 05-180 | |
| Na2HPO4 | EMD Millipore | SX-0720-1 | use to make 10X PBS stock |
| NaCl | RPI | S23020 | use to make 10X PBS stock and borate buffer |
| NaOH (1 N) | Sigma-Aldrich | S2770 | dilute with ultrapure water to 0.1 N |
| Nu-Serum (low-protein serum) | BD Biosciences | 355500 | use to make invadopodia medium |
| Paraformaldehyde | Acros | 416785000 | typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl) |
| PBS (sterile) | Cellgro | 21-040-CV | use for cell culture |
| RPMI 1640 | Cellgro | 10-040-CV | use to make invadopodia medium |
| Sodium borohydride | Sigma-Aldrich | 452882 | prepare fresh in fume hood 1 mg/ml in 1X PBS |
| sodium metaborate tetrahydrate | Sigma-Aldrich | S0251 | use to make borate buffer |
| Sucrose | RPI | S24060 | typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 °C (preheat PBS to dissolve gelatin easily) |
| TEMED | Bio-Rad | 161-0800 | |
| Triton X-100 | Alfa Aesar | A16046 | make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining |
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Diesen Artikel zitieren
Jerrell, R. J., Parekh, A. Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells. J. Vis. Exp. (95), e52343, doi:10.3791/52343 (2015).