研究使用细胞外基体水凝胶的正常组织辐射效应
Summary
该协议提出了一种在外体照射后,对母体乳腺脂肪垫进行去细胞化和随后水凝胶形成的方法。
Abstract
辐射是治疗三阴性乳腺癌患者的一种疗法。辐射对健康乳腺组织细胞外基质(ECM)的影响及其在原发性肿瘤部位局部复发中的作用尚不得而知。在这里,我们提出了一种从母体乳腺脂肪垫中提取的ECM水凝胶的去细胞化、冻干和制造方法。对脱细胞化过程的有效性进行了评价,并评估了变菌学参数。GFP-和荧光素酶标记的乳腺癌细胞封装在水凝胶中,表明辐照水凝胶的增殖增加。最后,利用phalloidin结合染色来可视化封装肿瘤细胞的细胞骨架组织。我们的目标是提出一种用于体外研究的培养水凝胶的方法,模拟体内乳房组织环境及其对辐射的反应,以研究肿瘤细胞行为。
Introduction
癌症的特点是细胞的过度增殖,可以避免凋亡,也可以转移到遥远的站点1。乳腺癌是美国女性最常见的发病之一,2018年估计有26.6万新发病例和4万例死亡。特别具有攻击性和难以治疗的亚型是三阴性乳腺癌(TNBC),它缺乏雌激素受体(ER)、孕激素受体(PR)和人类表皮生长因子(HER2)。放射治疗通常用于乳腺癌,以消除肿瘤切除术后的残余肿瘤细胞,但超过13%的TNBC患者在原发性肿瘤部位3仍然出现复发。
众所周知,放射治疗是有效的缓解转移和复发,因为块切除术和辐射的组合导致相同的长期生存,作为乳房切除术4。然而,最近已经表明,放射治疗与免疫功能低下设置5、6的原发性肿瘤部位局部复发有关。此外,众所周知,辐射通过诱导纤维化7改变正常组织的细胞外基质(ECM)。因此,了解辐射引起的ECM变化在控制肿瘤细胞行为中的作用是很重要的。
脱细胞组织已被用作体外模型来研究疾病8,9。这些去细胞化组织可保存ECM成分,并重述体内复合体ECM。这种脱细胞组织ECM可以进一步处理和消化,形成重组的ECM水凝胶,可用于研究细胞生长和功能10,11。例如,从脱细胞的人类脂吸剂和心肌组织中提取的可注射水凝胶作为组织工程的非侵入性方法,而从猪肺组织中提取的水凝胶被用作体外检测方法中生干细胞附着和活力12,13,14。然而,正常组织辐射损伤对ECM特性的影响尚未研究。
从ECM衍生的氢凝胶对体外体外现象的研究潜力最大。其他几种材料已经研究过,包括胶原蛋白、纤维蛋白和母蛋白,但很难综合地概括ECM13的成分。使用ECM衍生水凝胶的一个优点是ECM含有特定组织所需的蛋白质和生长因子14,15。在块切除术期间对正常组织的照射会导致ECM的显著变化,ECM衍生的水凝胶可用于在体外研究这种效应。这种方法可能导致更复杂和更准确的疾病体外模型。
在这项研究中,我们让母体乳腺脂肪垫(MFP)受到外体辐射。MFP被脱细胞制成预凝胶溶液。水凝胶是由嵌入的4T1细胞,一个小鼠TNBC细胞系形成的。研究了水凝胶材料的脑学特性,并在水凝胶内评估了肿瘤细胞动力学。由辐照的MFP制成的水凝胶增强了肿瘤细胞增殖。未来的研究将纳入其他细胞类型,以研究治疗后癌症复发背景下的细胞-细胞相互作用。
Protocol
动物研究是根据范德比尔特大学机构动物护理和使用委员会批准的机构准则和协议进行的。 1. MFP的准备和外生辐照 使用CO2窒息和宫颈脱位牺牲贫血Nu/Nu小鼠(8-10周)。 使用 70% 乙醇清洁皮肤。 使用预消毒剪刀和钳子从牺牲的小鼠身上收集乳腺脂肪垫 (MFP),在含有完整 RPMI 介质的 15 mL 锥形管中(RPMI 补充 1% 青霉素-链霉素和 10% 胎儿牛血清)(参见?…
Representative Results
使用图1A所示的程序在辐照后对MFP进行去细胞化。图为MFP预脱细胞化(图1B)和脱细胞后(图1C)。使用血氧素和欧辛(H & E)染色确认去细胞化,并使用1-([4-(Xlylazo)xlyl_azo-2-纳普霍尔染色法评估脂质含量(图2)。ECM水凝胶的学性质在37°C下也得到评估(图3)。在所有条件下,储存模量均高于损耗模量,表明水凝胶形成稳定。 <…
Discussion
这种水凝胶形成方法很大程度上取决于起始组织的数量。Murine MM 非常小,脱细胞化过程可显著减少材料(表 1)。该过程可以重复与更多的多功能一机,以提高最终产量。铣削是另一个可能导致材料损失的重要步骤。其他人已经展示了成功的低温磨机,但该协议是基于铣削通过手持砂浆和电钻与刺带附件8,17。这具有降低资本成本和最小化材料损失…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Laura L. Bronsart博士提供GFP和荧光素酶-4T1细胞,Edward L. LaGory博士就1-([4-(Xlylazo)xlyl_azo]-2-脱脂醇染色提供建议,克雷格·杜瓦尔博士为IVIS和冻合剂使用,斯科特·盖尔克博士为流流质表提供建议使用。这项研究得到了NIH资助#R00CA201304资助。
Materials
| 10% Neutral Buffered Formalin, Cube with Spigot | VWR | 16004-128 | – |
| 2-methylbutane | Alfa Aesar | 19387 | – |
| AR 2000ex Rheometer | TA Instruments | 10D4335 | rheometer |
| Bovine Serum Albumin | Sigma-Aldrich | A1933-25G | – |
| calcein acetoxymethyl (calcein AM) | Molecular Probes, Inc. | C1430 | – |
| D-Luciferin Firefly, potassium salt | Biosynth Chemistry & Biology | L-8820 | (S)-4,5-Dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazolecarboxylic acid potassium salt |
| DPX Mountant for Histology | Sigma-Aldrich | 06522-500ML | – |
| Dulbecco's phosphate-buffered saline | Gibco | 14040133 | – |
| Eosin-Y with Phloxine | Richard-Allan Scientific | 71304 | eosin |
| ethidium homodimer | Molecular Probes, Inc. | E1169 | ethidium homodimer-1 (EthD-1) |
| Fetal Bovine Serum | Sigma-Aldrich | F0926-500ML | – |
| Fisher Healthcare Tissue-Plus O.C.T. Compound | Fisher Scientific | 23-730-571 | cryostat embedding medium |
| Fluoromount-G | SouthernBiotech | 0100-01 | aqueous based mounting medium |
| FreeZone 4.5 | Labconco | 7751020 | lyophilizer |
| Hoechst 33342 Solution (20 mM) | Thermo Scientific | 62249 | blue fluorescent dye |
| Hydrochloric acid | Sigma-Aldrich | 258148-500ML | – |
| IVIS Lumina III | PerkinElmer | CLS136334 | bioluminescence imaging system |
| Kimtech Science Kimwipes | Kimberly Clark | delicate task wipes | |
| n-Propanol (Peroxide-Free/Sequencing), Fisher BioReagents | Fisher Scientific | BP1130-500 | – |
| Oil Red O | Sigma-Aldrich | O0625-25G | 1-([4-(Xylylazo)xylyl]azo)-2-naphthol |
| OPS Diagnostics CryoGrinder | OPS Diagnostics, LLC | CG-08-02 | – |
| PBS (10X), pH 7.4 | Quality Biological, Inc. | 119-069-151 | Phosphate-buffered saline |
| Penicillin-Streptomycin | Gibco | 15140-122 | – |
| Pepsin from porcine gastric mucosa | Sigma-Aldrich | P6887-5G | pepsin |
| Peracetic acid | Sigma-Aldrich | 77240-100ML | – |
| Phalloidin-iFluor 594 Reagent (ab176757) | abcam | ab176757 | phalloidin conjugate |
| Propylene glycol | Sigma-Aldrich | W294004-1KG-K | – |
| Richard-Allan Scientific Signature Series Bluing Reagent | Richard-Allan Scientific | 7301L | bluing agent |
| Richard-Allan Scientific Signature Series Hematoxylin 7211 | Richard-Allan Scientific | 7211 | – |
| RPMI Medium 1640 | Gibco | 11875-093 | – |
| Sodium deoxycholate, 98% | Frontier Scientific | JK559522 | deoxycholic acid |
| Sucrose | Sigma-Aldrich | S5016 | – |
| Triton x-100 | Sigma-Aldrich | X100-100ML | t-Octylphenoxypolyethoxyethanol |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200-056 | – |
| Whatman qualitative filter paper, Grade 4 | Whatman | 1004-110 | grade 4 qualitative filter paper |
| Xylenes (Certified ACS), Fisher Chemical | Fisher Scientific | X5-4 | – |
References
- Hanahan, D., Weinberg, R. A. The hallmarks of cancer. Cell. 100 (1), 57-70 (2000).
- Siegel, R. L., Miller, K. D., Jemal, A. Cancer statistics, 2018. CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).
- Lowery, A. J., Kell, M. R., Glynn, R. W., Kerin, M. J., Sweeney, K. J. Locoregional recurrence after breast cancer surgery: a systematic review by receptor phenotype. Breast Cancer Research and Treatment. 133 (3), 831-841 (2012).
- Miller, K. D., et al. Cancer treatment and survivorship statistics, 2016. CA: A Cancer Journal for Clinicians. 66 (4), 271-289 (2016).
- Vilalta, M., Rafat, M., Giaccia, A. J., Graves, E. E. Recruitment of Circulating Breast Cancer Cells Is Stimulated by Radiotherapy. Cell Reports. 8 (2), 402-409 (2014).
- Rafat, M., Aguilera, T. A., Vilalta, M., Bronsart, L. L., Soto, L. A., von Eyben, R., Golla, M. A., Ahrari, Y., Melemenidis, S., Afghahi, A., Jenkins, M. J., Kurian, A. W., Horst, K. C., Giaccia, A. J., Graves, E. E. Macrophages Promote Circulating Tumor Cell-Mediated Local Recurrence Following Radiation Therapy in Immunosuppressed Patients. Cancer Res. 75 (15), 4241-4252 (2018).
- Haubner, F., Ohmann, E., Pohl, F., Strutz, J., Gassner, H. G. Wound healing after radiation therapy: Review of the literature. Radiation Oncology. 7 (1), 1-9 (2012).
- Beachley, V. Z., et al. Tissue matrix arrays for high throughput screening and systems analysis of cell function. Nature Methods. 12 (12), 1197-1204 (2015).
- Tian, X., et al. Organ-specific metastases obtained by culturing colorectal cancer cells on tissue-specific decellularized scaffolds. Nature Biomedical Engineering. 2 (6), 443-452 (2018).
- Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., Badylak, S. F. Extracellular matrix hydrogels from decellularized tissues: Structure and function. Acta Biomaterialia. 49, 1-15 (2017).
- Hinderer, S., Layland, S. L., Schenke-Layland, K. ECM and ECM-like materials — Biomaterials for applications in regenerative medicine and cancer therapy. Advanced Drug Delivery Reviews. 97, 260-269 (2016).
- Young, D. A., Ibrahim, D. O., Hu, D., Christman, K. L. Injectable hydrogel scaffold from decellularized human lipoaspirate. Acta Biomaterialia. 7 (3), 1040-1049 (2011).
- Singelyn, J. M., Christman, K. L., Littlefield, R. B., Schup-Magoffin, P. J., DeQuach, J. A., Seif-Naraghi, S. B. Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. Biomaterials. 30 (29), 5409-5416 (2009).
- Pouliot, R. A., et al. Development and characterization of a naturally derived lung extracellular matrix hydrogel. Journal of Biomedical Materials Research Part A. 104 (8), 1922-1935 (2016).
- Bonvillain, R. W., et al. A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells. Tissue Engineering Part A. 18 (23-24), 2437-2452 (2012).
- Brown, B. N., et al. Comparison of Three Methods for the Derivation of a Biologic Scaffold Composed of Adipose Tissue Extracellular Matrix. Tissue Engineering Part C: Methods. 17 (4), 411-421 (2011).
- Link, P. A., Pouliot, R. A., Mikhaiel, N. S., Young, B. M., Heise, R. L. Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture. Journal of Visualized Experiments. (119), 1-9 (2017).
- Massensini, A. R., et al. Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity. Acta Biomaterialia. 27, 116-130 (2015).
- Mierke, C. T., Frey, B., Fellner, M., Herrmann, M., Fabry, B. Integrin 5 1 facilitates cancer cell invasion through enhanced contractile forces. Journal of Cell Science. 124 (3), 369-383 (2011).
- Ahmadzadeh, H., et al. Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion. Proceedings of the National Academy of Sciences. 114 (9), E1617-E1626 (2017).
Cite This Article
Alves, S. M., Zhu, T., Shostak, A., Rossen, N. S., Rafat, M. Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels. J. Vis. Exp. (149), e59304, doi:10.3791/59304 (2019).