作者要感谢biolitec研究有限公司（德国耶拿）对好心为我们提供了脂质体mTHPC配方，并特别感谢苏珊娜格拉夫和阿诺Wiehe工厂对他们的帮助和技术专长。我们还要感谢凯尔斯丁里迪，谁产生的结果有很大一部分，是coresponsible其出版11。 这项工作是由来自Schweizerischer社团组织Balgrist，苏黎世大学，苏黎世Krebsliga以及从沃尔特湖和约翰娜·沃尔夫基金会，瑞士苏黎世的赠款，并从EuroNanoMed ERA-NET / SNF瑞士拨款补助支持美国国家科学基金会31NM30-一分之十三万一千零四。这项工作也是由HSM的坎顿苏黎世的肌肉骨骼肿瘤（高度专业化医学）计划的支持。

1。 mTHPC在相应的低的吸收和高转移居屋和143B的OS细胞系的比较<ol><li>制备含有DMEM，含F12，并且在一个比4.5:4.5:1热灭活的胎牛血清的细胞培养基。 注意:在PS mTHPC的脂质体制剂溶解在水中以1.5毫克/毫升mTHPC的最终浓度。 </li><li>板0.2×10 6个居屋和143B细胞/孔在6孔板中（一式三份对各条件），并让细胞贴壁过夜。 注意:该数量必须调节到个体的细胞系。 80-90％A汇合是在实验时最合适。 </li><li>在接下来的一天，孵育细胞mTHPC固定浓度不同的时间段（时间依赖性摄取），或者与在一个固定的时间周期（剂量依赖性摄取）增加mTHPC的浓度。 </li><li>孵育细胞mTHPC具体时间Points或mTHPC的浓度。 注:在本研究中，居屋和143B细胞系孵育0.6微克/毫升mTHPC为0，2.5，5，或10小时，或用0，0.6，2.5和10微克/毫升mTHPC 5小时。所施加的时间周期和剂量可能取决于所使用的细胞株不同而不同。 </li><li>记住，总是在黑暗或暗光条件下工作，防止细胞的直接曝光。 </li><li>将细胞在指定的条件下孵育后，吸出培养基，用PBS洗细胞3次。接着，取下电池用0.5ml胰蛋白酶/ EDTA和计数细胞。 </li><li>脱离后，离心细胞，在400×g离心5分钟，吸出培养基，重悬细胞于PBS中，以200,000细胞/ ml的终密度。 </li><li>移液管将100μl所获得的细胞悬浮液（ 即 20,000个细胞）在96孔板中，并测量这些细胞在荧光分光光度计的荧光。设置S表示mTHPC荧光测量:417 nm处的激发和652 nm处的发射。 </li><li>为了计算每个细胞的内化mTHPC的量，归一化的相对荧光单位（RFU）的细胞数目和细胞的体积，如下所示: </li><li>从0-4微克/毫升（使用一式三份测量）使用不同浓度的mTHPC在PBS中制备的标准曲线。吸取100微升每种稀释液在96孔板中，并测量使用根据步骤1.6中描述的设置的孔的荧光。 </li><li>通过mTHPC，这给mTHPC的浓度（以μg/ ml）的中井的线性标准曲线的斜率除以RFU（为20,000个细胞获得）。 </li><li>除以10这mTHPC浓度得到mTHPC在一个很好的总量（以μg，相当于100微升细胞悬浮液），并通过20,000个细胞的体积除以该值。 </li><li>计算的公式计算，一个球体的体积:（4/3×π）×3条规则。通过测量20个胰蛋白酶消化细胞的直径，在显微镜下，并除以2这个值取得的半径 r。 </li><li>最后，规范所有的值对那些10小时培养（剂量依赖性摄取）或10微克/毫升mTHPC（随时间变化的吸收），这是设置为100％，处理后得到的HOS细胞。 </li></ol> 2。 PS 体外的光毒性的测定<ol><li>种子3000个细胞/孔在96孔板（一式三份对mTHPC的每个浓度使用），并让他们附着过夜。同样准备一个额外的96孔板中，如下所述处理细胞，但随后把它们放在一边的相位衬度成像。 注意:数字必须调整每个单独的细胞系。 50-60％在实验时的汇合是理想的。 <ol><li>记住要添加单元格对应的暗毒性控制每个mTHPC浓度。该本身是细胞，将在选定的mTHPC浓度的存在或不存在下温育，但不会被点亮。 </li></ol></li><li>在接下来的一天，孵育细胞取决于细胞系不同mTHPC（0，0.0001，0.001，0.01，0.03，0.075，0.15，0.3，0.6，1.25，2.5，5和10微克/毫升）的浓度和让他们在黑暗中5小时。 <ol><li>参见1.3.2节 注意:孵育时间前照明依赖于细胞系和所使用的PS，因此最好是在单独的实验中，测试不同浓度和不同的时间点。 </li></ol></li><li>在温育指定的时间内，用PBS洗细胞两次，并没有mTHPC添加新的新鲜培养基中。 </li><li>照亮细胞用652纳米二极管激光器，具体为mTHPC吸收光谱（ 见图1）。 <ol><li>激光设置为21.88毫瓦/厘米2的电源在13.5厘米DISTA的高度考从细胞中，以获得5焦耳/厘米2的能量剂量。照亮细胞为230秒。不亮的暗毒性对照细胞（有，无mTHPC）。 注意:一定要戴眼镜以保护眼睛免受用于PS的激发激光。 </li></ol></li><li>光照后，保持细胞在黑暗中，在37℃24小时。随后，添加水溶性四氮唑（WST-1）试剂（10μl/100微升培养基）。 </li><li>在加入WST-1试剂后三小时，测量96孔板415nm处的各个孔的吸光度。 </li><li>为了计算存活细胞的百分比，设定为无mTHPC处理的细胞为每个条件为100％（ 即照明系统，无mTHPC和nonilluminated，没有mTHPC）中得到的值的平均值。 </li></ol> 3。的细胞数量估计由细胞计数<ol><li>种子20,000个细胞/我们LL在24孔板中，并让他们坚持过夜。 </li><li>于室温孵育5小时以mTHPC（0.001，0.003，0.15，0.6和1.25微克/毫升），并如上所述照射它们。 </li><li>收集培养基，一旦用PBS以400×g离心洗细胞，胰蛋白酶消化它们和将它们收集通过离心分离5分钟。 </li><li>离心后，吸出培养基并悬浮细胞于200μl的新鲜培养基。 </li><li>细胞计数在纽鲍尔室。 </li></ol>

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作者宣称没有竞争的经济利益或利益冲突。

为了实现在响应PDT最佳的细胞毒性，关键是要选择合适的激光设置和孵育时间。这里所描述的过程是一致的，高效的，确定的PS吸收和量化PDT诱导的细胞毒性体外 。使用PS mTHPC的特定吸收波长，蜂窝PS摄取可以以直接的方式来确定，并且PS可以被激活，以产生细胞毒性的活性氧物种。 利用这种体外设置中，所有的参数，如聚苯乙烯浓度，药物的光的时间间隔和激?...

骨肉瘤（OS）的艺术处理今天的状态，原发性骨肿瘤，包括为新辅助化疗和手术的组合。这个治疗方案显示增加的患者从使用化疗前约20％的局部疾病的存活率，到目前的60-70％3,4。然而，在过去的二十年中，操作系统患者局部病变的总生存期已趋于稳定4，5。此外，这些患者中30％-40％在确诊后3年内复发和转移性疾病继续有20-30％4一个贫穷的生存，6，7。为了改善这些患者的预后，新的治疗策略需要发展。 光动力疗法（PDT），一个相当新颖的抗癌疗法，采用特定波长光的一个photosensi的激励光敏剂（PS），其中其注射到血液中后积聚在肿瘤细胞。在PS的激光激发产生的氧自由基在氧的存在下，其诱导肿瘤细胞和细胞死亡的细胞毒反应。除了这个主要机制，另外两个PDT诱发的生物学过程有助于减少肿瘤的生长:PDT引起血管收缩和血栓形成的肿瘤微血管，因而局部缺氧及缺氧的肿瘤内，导致肿瘤梗死。最后，PDT受伤和死亡的肿瘤细胞引发局部免疫反应，一个很独特的PDT的功能。这涉及补体系统和抗原的活化呈递树突状细胞8。因此，条件是肿瘤抗原与淋巴样细胞，随后激活的演示文稿中创建，导致肿瘤特异性免疫。 到目前为止，PDT已被用于治疗多种类型的软组织肿瘤和hyperpl亚洲，如光化性角化病，Barrett食管，支气管内肿瘤，膀胱癌，基底细胞癌，和姑息治疗头颈部癌2。治疗是已知的诱导局部，大规模坏死，只有很少的副作用，因此具有选择性地根除肿瘤组织的潜力。尽管有这些优势，PDT的应用程序保持技术上比化疗药物的管理更加严格。为了达到最大的效力，在PS的浓度，光照射时间和总的光能量传递需要被优化。这可以在体内实验来完成，但是，由于相对大量的需要被优化的参数的初步确定体外最佳条件下，它是更有效的。 在下面描述的实验中，我们使用PS 5,10,15,20 -四（间-羟基苯基）叶绿素测试PDT的体外功效奥林简称mTHPC（ 图1A）。 mTHPC是在医药品Foscan，这是目前在临床上对姑息治疗头部和颈部癌症的使用的活性物质。它是最有效的PS中的一个，在低浓度下已经大规模诱导的细胞损伤，并且它被证明是优于其它PS在组织穿透9,10的条款。它的光吸收光谱（ 图1B）示出了两个显着的峰，一个在417 nm和第二在652纳米，这是用于积累PS的组织定位和对PDT诱导，分别。 目前，脂质体制剂用于mTHPC正在开发中。在这里，我们描述的程序来量化这种脂质体制剂的摄取，并且在两个人的OS细胞株进行PDT;低转移性居屋和高转移性143B细胞。一些这里介绍的数据已报告前面11。钍这里描述É方法让我们来研究PDT疗效转移表型的影响。 143B细胞原位注射到免疫缺陷的SCID小鼠的后肢引起intratibial转移性原发性肿瘤，模型密切模仿人类转移性疾病。因此，建议在体外实验中是完全合适的，以评估在体内实验中用于以后的最佳PDT设置。

<table><tbody><tr><td>DMEM</td><td>PAA GmbH, Freiburg, Germany</td><td>E15-883</td><td></td></tr><tr><td>HAM F12</td><td>PAA GmbH, Freiburg, Germany</td><td>E15-817</td><td></td></tr><tr><td>Heat-inactivated fetal calf serum</td><td>GIBCO, Basel, Switzerland</td><td>10500-064</td><td></td></tr><tr><td>mTHPC</td><td>biolitec Research GmbH, Jena, Germany</td><td></td><td>As this liposomal formulation is originating from R&amp;amp;D&lt;br/&gt;no catalogue number is available at the moment. Stock: 1.5 mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &gt;99% purity)</td></tr><tr><td>Spectramax Gemini XS plate reader</td><td>Molecular Devices, Sunnyvale, CA</td><td>ID# 861</td><td></td></tr><tr><td>Microscope Zeiss Observer.Z1</td><td>Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td><td></td><td></td></tr><tr><td>Ceralas PDT 652 nm Laser</td><td>biolitec AG, Jena, Germany</td><td>LD652nm2W400u</td><td></td></tr><tr><td>Water-soluble tetrazolium (WST) reagent</td><td>Roche Diagnostics AG, Rotkreuz, Switzerland</td><td>1644807</td><td></td></tr></tbody></table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

近年来，出现了在寻找更有效的治疗对癌症具有较少的全身性副作用的困难。因此，光动力疗法是一种新的方法对多种肿瘤有选择性的治疗。 光动力疗法（PDT），使一个无毒的光敏剂（PS），其中，经活化的光在氧的存在下的特定波长的光，产生氧自由基，引发细胞毒性反应1的使用。尽管其批准几乎是二十年前由美国FDA，PDT是时下唯一用于治疗癌症类型（皮肤，膀胱）和nononcological疾病（牛皮癣，光化性角化病）2的数量有限。 利用光动力疗法的主要优点是能够执行一个局部治疗，以防止全身性副作用的能力。此外，它可以让肿瘤在细腻的部位治疗（ 如周围神经或血管）。这里，一个intraoperatPDT的IVE应用被认为是在骨肉瘤（OS），肿瘤的骨，针对原发肿瘤的卫星遗留在肿瘤外科手术切除肿瘤后，周围的组织。治疗的目标是减少复发的数量以及减少为（术后）转移的风险。 在本研究中，我们提出在体外 PDT程序以建立有效的治疗方法，用于重现人类疾病中确立intratibial OS的小鼠模型中广泛使用的操作系统的细胞系的最优PDT设置。在PS mTHPC的吸收进行了检查用分光光度计和光毒性被挑起与mTHPC的激光激发在652 nm到诱导细胞死亡评估用WST-1法和存活细胞计数。所建立的技术使我们能够确定最佳PDT设置在动物模型中未来的研究。他们是一个简单而快速的工具，用于PDT的疗效评价<em&gt;在体外体内应用程序之前。

与此报告的技术，我们研究mTHPC为基础的PDT在人类OS细胞。首先，mTHPC的时间和剂量依赖性摄取研究在低转移性居屋和在高转移性143B OS细胞系。 mTHPC摄取可以通过测量mTHPC的荧光用荧光分光光度计（ 图2中 ，再现从雷迪等人 11的许可）进行评估。 图2A示出mTHPC的时间依赖性摄取。将细胞悬浮液的荧光强度表示mTHPC的胞内水平。 图2B示出了剂量依赖?...

Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

这里所报告的协议允许光动力疗法（PDT）中的细胞系和的PDT设置施加治疗在动物模型中之前，优化的效果进行评价。

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

cytotoxic-efficacy-photodynamic-therapy-osteosarcoma-cells

Research

JoVE Journal

Medicine

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

12.8K 次观看.  Balgrist University Hospital, Zurich, Switzerland. 这里所报告的协议允许光动力疗法（PDT）中的细胞系和的PDT设置施加治疗在动物模型中之前，优化的效果进行评价。 

视频: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

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.

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. ...

科研

癌症研究

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

化学

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

<meta charset="utf8"></head><body>骨肉瘤 PDX 小鼠模型的方案：研究生长和耐药性

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

<meta charset="utf8"></head><body>作者聚焦：在免疫缺陷小鼠中复制人骨肉瘤进展用于癌症研究

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

生物化学

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

光动力疗法治疗骨肉瘤细胞的细胞毒功效

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此视频中的章节

光动力疗法的抗癌功效肺癌靶向纳米粒子

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物