这项研究是由赠款没有支持。 14-2014-017从SNUBH研究基金。 该作者感谢J.帕特里克·巴伦，名誉教授，日本东京医科大学兼职教授，首尔国立大学盆唐医院这个手稿他的无偿编辑。

注:所有动物研究方案是由首尔国立大学盆唐医院的机构动物护理和使用委员会（BA1308-134 / 072-01）的批准。 1.玻尿酸，神经酰胺的合成（HACE） <ol><li>在60的双蒸水（DDW）毫升溶解12.21毫摩尔透明质酸（HA）寡聚物和9.77毫摩尔四- 正丁基氢氧化铵（TBA）的。搅拌30分钟。 </li><li>以合成的DS-Y30连接体，溶解8.59毫摩尔的DS-Y30神经酰胺和9.45毫摩尔的三乙胺在25毫升四氢呋喃（THF）。用8.59毫摩尔的THF 4-氯甲基苯甲酰氯混合。搅拌在60℃下6小时。 </li><li>溶解所合成的8.10毫摩尔的HA-TBA和0.41毫摩尔的DS-Y30接头中和THF的混合物中的乙腈（4:1，体积/体积）。搅拌在40℃5小时。 </li><li>通过与过滤剂过滤除去杂质，并消除由真空蒸发有机溶剂。净化用渗析膜产物（分子量截止:3.5 kDa的），并冷冻干燥。 </li></ol> 2.纳米颗粒的制备<ol><li>溶解1毫克的HB和在0.5ml二甲基亚砜（DMSO）的1毫克的紫杉醇和通过涡旋混合5分钟，用0.5毫升DDW的混合。然后，溶解HACE在通过涡旋混合该混合物另外5分钟。 </li><li>为了消除在氮气缓流在70℃下4小时的溶剂，热。 </li><li>悬浮脑水肿，HB组成的薄膜，紫杉醇用1毫升DDW的。用注射器过滤器（0.45微米孔径大小）过滤以除去未包封的药物。 </li></ol> 3. 在体外光毒性<ol><li>在肺癌细胞系中的纳米颗粒的摄取<ol><li>制备的RPMI-1640含10％中等（体积/体积）胎牛血清和1％（重量/体积）青霉素 - 链霉素。 </li><li>种子A549细胞在24孔细胞培养板中以1×10 5个细胞的密度/孔（一式三份对各组）。在湿润的5％CO 2和95％空气气氛中孵育在37℃下24小时。 </li><li>细胞附着后，除去培养基并加入1 ml磷酸盐缓冲盐水（PBS）洗细胞。 </li><li>溶解在PBS中，纳米颗粒为2μM/ ml的HB的终浓度。然后，孵育将细胞用1ml的PBS，空纳米颗粒，HB-纳米粒，或HB-P-纳米粒的各孔在黑暗中4小时。 </li><li>除去所有溶液，并通过加入1ml冷PBS洗细胞。重复洗涤步骤一次。添加新鲜的培养基。 </li></ol></li><li>细胞生存力测定法<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜，并与在黑暗中的不同时间一个PDT激光器（630纳米，400毫瓦/厘米2）照射细胞:0，5，10，20，30，和40秒（0，2，4 ，8，12，和16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>添加10微升的细胞毒性测量溶液每个孔中。孵育在黑暗中搅拌2小时。 </li><li>用酶标仪测量450nm处的吸光度。 </li></ol></li><li>显微分析<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜和照亮与在黑暗中的PDT激光器（630纳米，400毫瓦/厘米2）的细胞为0，20，或40秒（0，1,8或16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>添加膜联蛋白V-FITC的50微升和50微升4&#39;，6-二脒基-2-苯基吲哚（DAPI，1.5微克/毫升）的。轻轻摇动板和孵化它在黑暗中室温下15分钟。 注:使用膜联蛋白V-FITC从荧光显微镜套件。 </li><li>通过加入1ml冷PBS洗细胞。重复洗涤步骤一次。保持所述细胞在新鲜的PBS中。确定用光学显微镜放大100倍的凋亡细胞。 </li></ol></li><li>荧光激活细胞分选（FACS）分析<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜和照亮用PDT激光（630纳米，400毫瓦/厘米2）的细胞为0，20，或40秒（0，1,8或16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>悬浮细胞在1ml 1×结合缓冲液（稀释10倍的结合缓冲液的1份; 0.1M HEPES / NaOH水溶液（pH为7.4），1.4M的NaCl和25mM的氯化钙2至9份蒸馏水）和转让100微升样品溶液至5毫升培养管。 </li><li>添加膜联蛋白V-FITC的5和5μl碘化丙啶（PI）的。轻轻涡旋管孵育在黑暗中室温（RT）15分钟。添加400μl的1×结合缓冲液。 注:从荧光显微镜套件使用膜联蛋白V-FITC和PI。 </li><li>通过使用FACS 14识别凋亡细胞。 PI和膜联蛋白V-FITC的激发激光线分别是488纳米和635纳米。测量PI和膜联蛋白V-FITC的荧光发射在610±20纳米和660±20纳米，分别。收集每10,000事件流式细胞仪获得的细胞。 </li></ol></li></ol>在荷瘤小鼠4. 在体内的抗癌功效<ol><li>肺癌引起的小鼠模型<ol><li>制备1×10 6个 A549细胞在0.1ml RPMI-1640培养基;保持它在冰上。 </li><li>用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮（1的混合物麻醉小鼠:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>注入细胞皮下到BALB / c雄性裸鼠的左侧面（6 - 7周龄，20 - 22克）。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。 注:直到它重新获得足够的意识，以保持胸骨斜卧不要离开无人看管的动物。不要返回已动过手术，以公司的其他动物，直到它已经完全康复的动物。保持无特定病原体（SPF）的条件下的小鼠。 </li><li>每天测量肿瘤大小与卡钳。计算肿瘤体积（毫米3）为（长×宽2）/ 2，当肿瘤大小在体积达到约200mm 3，开始实验。 </LI&gt; </ol></li><li>抗癌功效研究<ol><li>随机划分小鼠分成4组（n = 10每组）。 </li><li>麻醉小鼠用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮的混合物（1:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>溶解在PBS中，纳米颗粒为2毫克/毫升的HB的终浓度。通过尾静脉（2毫克/公斤为HB）第0天及7两次注射的PBS，无HB，HB-纳米粒，或HB-P-纳米粒。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。 注:直到它重新获得足够的意识，以保持胸骨斜卧不要离开无人看管的动物。不要返回已动过手术公司其他动物直至完全的动物恢复。保持笼黑暗和具体的SPF条件下。 </li><li>每次注射后24小时，麻醉小鼠用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮的混合物（1:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>放置PDT纤维下的肿瘤部位（以1厘米的距离从PDT光纤到肿瘤）。戴激光安全眼镜，关掉开关，并用PDT激光（630纳米，400毫瓦/厘米2）500秒（200焦耳/厘米2）1和8天两次照射肿瘤。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。不要返回具有undergon动物Ë手术给公司其他动物，直到完全康复。 </li><li>保持SPF条件下的笼子里。保持在黑暗中的笼激光治疗后24小时。 </li><li>直观地监视肿瘤体积，并在每天肿瘤部位的变化。测量肿瘤大小用卡尺，并计算出体积为（长×宽2）/ 2（毫米3）。就拿肿瘤部位的照片，每天检查后PDT肿瘤表面的改变。 </li><li>在第16天，通过使用异氟烷终端麻醉牺牲每组五只小鼠。 </li><li>随着镊子和剪刀，剪开外皮和揭露肿瘤15。仔细收获他们。解决这些问题，在10％福尔马林，在石蜡中嵌入其中，和与用于组织学分析16苏木精和曙红（H＆E）染色它们。 </li><li>监测后45天，用异氟烷麻醉终端牺牲剩余的老鼠。 </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

在这项研究中最关键的步骤是选择合适的激光条件:波长，功率和照射时间。适合于特定的光敏剂的光的适当波长是必要的PDT。我们使用了一个630纳米的激光，是适当的竹红菌素B的输出功率是另一个重要的因素，这是基于许多试验性研究设定在400毫瓦/厘米2。输出功率超过400毫瓦/厘米2受损细胞或动物的皮肤表面由于照射本身，而低于400毫瓦/厘米2的输出功率太弱，以显示?...

Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4. 
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13. 
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2. 
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, HB) solution, photosensitizer-encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>oligo hyaluronic acid</td>
			<td>Bioland Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Y30 (ceramide 3B; mainly N-oleoyl-phytosphingosine)</td>
			<td>Doosan Biotech Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>adipic acid dihydrazide</td>
			<td>Sigma Aldrich</td>
			<td>A0638</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(chloromethyl)benzoyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>270784</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>T0546</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filter</td>
			<td>Sartorius Stedim Biotech GmbH</td>
			<td>17762</td>
			<td>15 mm, RC, PP, 0.45 &#181;m</td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-GeBAflex tubes</td>
			<td>Gene Bio-Application Ltd.</td>
			<td>D070-12-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>Taihua Corporations</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life Technologies, Inc.</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Celite (Filter agent)</td>
			<td>Sigma Aldrich</td>
			<td>6858</td>
			<td>See step 1.4</td>
		</tr>
	</tbody>
</table>

anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

我们制备的两个HB-NP和HB-P-纳米粒与上述技术。 HB-NP和HB-P-纳米粒子的平均粒径分别为220.9±3.2 nm和211.9±1.6纳米。 后用PBS，空纳米颗粒，HB-纳米粒，和HB-P-纳米粒孵育4小时，然后通过光照射（0至16焦耳/厘米2）A549细胞的细胞活力在图1中示出，如果没有光，则HB-NP治疗组无细胞毒性可言，而HB-P-NP-处理的细胞81....

Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted

Research

JoVE Journal

Cancer Research

8.9K 次观看.  Seoul National University Bundang Hospital. Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

视频: Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺肿瘤细胞招募分析

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

科研

癌症研究

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

建立和表征三个抗阿法替尼的肺腺癌PC-9细胞系与增加剂量的阿法替尼开发

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...