软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

摘要

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

摘要

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

引言

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell’s ability to proliferate and form colonies in suspension within a semi-solid agarose gel¹. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix².

A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.

PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination^3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers⁶. For example, we have previously demonstrated that a “pan-PADI” inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids⁶. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies⁷. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein⁸. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

研究方案

1.制备3％2-羟乙基琼脂糖

1. 到一个干净，干燥的100 ml玻璃瓶中加入0.9克2-羟乙基琼脂糖（琼脂糖VII），随后加入30ml蒸馏水。
2. 微波15秒，轻轻混合漩涡。重复此步骤至少三次，直到琼脂糖粉末完全溶解。
3. 高压釜中加入含有溶液的瓶子15分钟。
4. 允许琼脂糖解决方案，进一步使用前冷却至室温。存储溶液在RT。

2.编写底层:0.6％琼脂糖凝胶

1. 预暖几个5毫升和10ml移液器在37℃培养箱中，以防止琼脂糖从搬运时固化在吸管。
2. 部分地松开瓶盖和微波15秒的预制备3％2-羟乙基琼脂糖溶液。然后，轻轻地旋转解决方案和微波了15秒钟。注意:在纷飞的时候琼胶要小心本身的方案，因为当暴露于空气中，并可以溢出的溶液上升。
3. 如果存在残留的固体凝胶在瓶中，微波几秒钟。
4. 保持含在下一步骤，以防止该琼脂糖溶液从固化过早在45℃水浴琼脂糖溶液的瓶中。
5. 温馨MCF10DCIS媒体在37℃水浴。注意:MCF10DCIS媒体包括DMEM / F12，5％马血清，5％青霉素链霉素。
6. 转印3毫升使用预热的移液管的3％的琼脂糖溶液加入到无菌的50ml锥形管中。
7. 立即加列12毫升温MCF10DCIS媒体和轻轻颠倒锥形管混合与媒体的琼脂糖。尽量不形成任何气泡，因为它会与殖民地后计数干扰。
8. 轻轻加入2毫升该混合物到6孔培养板的各孔而不形成任何气泡。
9. 孵育的6孔培养板horizo​​ntaLLY在平坦表面上，在4℃下1小时，以允许混合物固化。
10. 混合物固化后，将板放入37℃培养箱中30分钟。底层是现在可以使用。

3.制备含细胞层:0.3％琼脂糖凝胶

1. Trypsinize MCF10DCIS细胞并稀释至4×10 ⁴个/ ml的细胞浓度。
2. 取2毫升使用预热移液器并传输3％琼脂糖的进入无菌50ml锥形管中。
3. 立即添加8毫升MCF10DCIS媒体到锥形管并轻轻颠倒混合与媒体的琼脂糖。避免形成任何气泡。
4. 取2毫升MCF10DCIS细胞（4×10 ⁴个/ ml）的中和治疗与BB-CLA（0μM（DMSO）或1μM的）。
5. 以1:1稀释，混合细胞与0.6％的琼脂糖。
6. 取1毫升细胞 - 琼脂糖混合物，轻轻加入到6孔培养p的底层晚期（2×10 ⁴个细胞/ ml）。
7. 水平放置的6孔培养板中在一个平面上，在4℃下至少15分钟，以允许在顶层固化。
8. 混合物固化后，将板放入37℃培养箱中培养一周增加馈送层之前。

4.准备饲养层的0.3％琼脂糖凝胶

1. 微波15秒的预制备3％2-羟乙基琼脂糖溶液。轻轻摇动解决方案和微波了15秒钟。
2. 平衡的琼脂糖溶液瓶在45℃水浴中。
3. 暖MCF10DCIS媒体在37℃水浴。
4. 混合1毫升3％的琼脂糖溶液用9ml温暖MCF10DCIS媒体到50ml锥形管中并轻轻颠倒混合与媒体的琼脂糖。避免形成气泡。
5. 治疗与BB-CLA的混合物（0μM（DMSO）或1μM的）。
6. 轻轻加1ml该混合物中（不包括用于明气泡）到含有底和软层中的6孔培养板的每个孔中。
7. 水平放置的6孔培养板中在一个平面上，在4℃下至少15分钟以使该混合物固化。
8. 饲养层固化后，将板放入37℃培养箱中。
9. 通过叠加将1ml 0.3％琼脂糖/中/处理溶液到现有的饲养层来补充细胞与新媒体直到集落形成观察到重复此馈送过程每周。注意:琼脂在柔软和饲养层是非常柔软的，因此，从饲养层添加的营养物将容易地扩散入含细胞层到达细胞。

5.数据收集

1. 2.5周细胞生长在软琼脂后，计数在使用光学显微镜每孔的集落数。为了便于定量，打印网格到透明度和电网连接到6孔板，以帮助找到那里的细胞计数中。自菌落大小（如量化由每个菌落的直径）将变化，预定义的参考菌落大小，以确定哪些集落进行评分。例如，包括70微米或在数据分析中较大的菌落尺寸。
2. 储存样品在4℃以防止进一步的集落形成，并为未来的计数。密封6孔培养板用封口膜，以防止凝胶干燥。

结果

软琼脂集落形成测定法可用于范围广泛的应用中记录癌细胞的致瘤性。这种技术的主要优点是，半固体基质选择性有利于细胞，它可以在一个锚定非依赖性方式增殖的生长。此特征主要表现由癌细胞而不由正常细胞。我们主要使用这种技术通过药物测试的肿瘤生长抑制的效果和测试对于过表达或我们的基因的兴趣，包括PADI基因的耗竭的效果，对乳腺癌细胞的致瘤性。这里，我们评估BB-CLA对的PADI2?...

讨论

的集落形成在软琼脂速率的变化取决于细胞类型^9。因此，细胞的数量，以开始与应优化，并相应地调整。一个建议的起始范围为每使用公一个6孔板在5×10 ^{2〜1×10 4}个细胞。此外，菌落大小取决于每个细胞的生长速度。因此，一个预定的截止菌落大小是需要的注释下游定量分析单个菌落。这里，菌落大于70微米的被量化，以避免包含非增殖细胞从最初的镀衍生的。

材料

Name	Company	Catalog Number	Comments
Zeiss Axiopot	Carl Zeiss Microscopy	1021859251
Inverted Microscope	Olympus	CKX41
DMEM/F-12	Lonza BioWhittaker	12-719F
HyClone Donor Equine Serum	Fisher Scientific	SH30074.03
Penicillin Streptomycin	Life Technologies	15140-122
2-Hydroxyethylagarose: Type VII, low gelling temperature	Sigma-Aldrich	39346-81-1