The use of photosensitizers to improve the efficiency of radiotherapy

Radiotherapy (RT) is currently the leading therapeutic strategy for prostate cancer (PCa) and a major curative treatment for head and neck squamous cell carcinoma (HNSCC) in combination with surgery or concurrent chemotherapy in advanced stages. Despite advances in radiotherapy leading to the precise delivery of high radiation doses, radioresistance and locoregional recurrence are the major cause of treatment failure. Photodynamic therapy (PDT) is safe, low toxic and selective method of cancer treatment. It utilizes non-invasive elements: photosensitizer (PS) and light of a specific wavelength that is able to photoactivate PS to generate cytotoxic reactive oxygen species (ROS). Highly penetrating X-ray during megavoltage clinical beam radiation can generate visible light in UVA spectrum and activate PSs placed in deep-seated tumors. Megavoltage irradiation can generate UVA light in two ways: 1) directly through photon that travels faster than the speed of light through a medium thereby generating Cherenkov light (called “radiotherapy enhanced with Cherenkov photo-activation” - RECA); 2) through nanoparticle scintillators that convert external X-ray photons to UV light (called X-PDT). X-ray induced PDT has become a promising, novel anti-cancer strategy. The aim of the project is to verify whether the use of PSs during clinical X-ray megavoltage beam irradiation increases the effectiveness of radiotherapy. In the project we will use drugs (ketoprofen, lomefloxacin, doxycycline, 8-methoxypsolaren) with proven phototoxic and anti-cancer properties. We will investigate whether they can serve as photosensitizers in RECA or X-PDT therapies. We will evaluate the phototoxic effect of increasing energy of megavoltage beam radiation (6 MV, 15 MV and 20 MV). Additionally, X-ray induced phototoxicity will be assessed following two irradiation regimens: single high dose irradiation (we will start from 10Gy and then the dose will be downsized to maintain the maximum benefit) or a fractionated dose (3x2.5Gy), which corresponds to the clinical hypofractionated radiotherapy regimen. For X-PDT yttrium oxide (Y2O3) loaded polymer nanospheres will be designed and constructed to serve as scintillator. Additionally, after determining the most efficient photosensitizer, we will construct Y2O3 and PSs co-loaded nanospheres. X-ray induced phototoxic potential of the tested drugs will be assessed against radioresistant HNSCC and PCa. Furthermore we will verify if radioresistant cells subjected to therapeutic regimen combining RT and PDT will be more sensitive to chemotherapeutics standardly used in the clinic and if small molecule inhibitors that target the survival or DNA repair pathways will increase the effectiveness of tested therapy. The efficiency of RECA or X-PDT therapies regimens will be assessed, among others by: examining the viability of cancer cells and the type of induced cell death, assessing ROS production and mitochondrial activity, radiosensitivity determination by colony-forming unit assay. The research hypothesis will be verified on mouse models of human HNSCC and PCa. The potential to trigger anti-tumor immune response and the role of immune cells in the investigated therapeutic regimen will be determined in immunocompetent mouse models of HNSCC and PCa. The project is innovative. Its aim is to develop a novel method of sensitizing cancer cells to radiotherapy using drugs with photosensitizing potential available in the clinic, which can significantly increase the effectiveness of radiotherapy in radiotherapy-resistant head and neck and prostate tumors.放疗（Radiotherapy, RT）目前是前列腺癌（prostate cancer, PCa）的主要治疗策略，也是晚期头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）联合手术或同步化疗的主要根治性治疗手段。尽管放疗技术的进步已实现高辐射剂量的精准递送，但辐射抵抗与局部区域复发仍是治疗失败的主要原因。 光动力疗法（photodynamic therapy, PDT）是一种安全、低毒且具有选择性的癌症治疗方法，其利用两类无创性要素：光敏剂（photosensitizer, PS）与特定波长的光线，后者可光活化光敏剂以产生具有细胞毒性的活性氧（reactive oxygen species, ROS）。临床使用的兆伏级射线照射（megavoltage clinical beam radiation）中，穿透力极强的X射线可在UVA光谱范围内产生可见光，并激活位于深部肿瘤内的光敏剂。兆伏级照射可通过两种方式产生UVA光：1）直接通过在介质中传播速度超过光速的光子产生切伦科夫光（Cherenkov light），该方法称为“切伦科夫光活化增强放疗”（RECA）；2）通过纳米粒子闪烁体将外部X射线光子转换为紫外光，该方法称为X-PDT。X射线诱导光动力疗法已成为一种极具前景的新型抗癌策略。 本项目的研究目的为验证在临床兆伏级X射线照射期间使用光敏剂是否可提升放疗的疗效。本项目将选用已被证实具有光毒性与抗癌特性的药物：酮洛芬（ketoprofen）、洛美沙星（lomefloxacin）、多西环素（doxycycline）、8-甲氧基补骨脂素（8-methoxypsolaren），探究其能否作为RECA或X-PDT疗法中的光敏剂。我们将评估不同能量的兆伏级射线照射（6 MV、15 MV与20 MV）的光毒性效应。此外，将通过两种照射方案评估X射线诱导的光毒性：单次高剂量照射（初始剂量为10 Gy，后续逐步下调剂量以实现最优疗效），或分割剂量照射（3次×2.5 Gy），后者与临床大分割放疗方案一致。针对X-PDT疗法，我们将设计并构建负载氧化钇（yttrium oxide, Y₂O₃）的聚合物纳米球作为闪烁体。在确定最优光敏剂后，我们还将构建同时负载Y₂O₃与光敏剂的纳米球。 我们将针对辐射抵抗性HNSCC与PCa细胞，评估受试药物的X射线诱导光毒性潜力。此外，我们将验证接受放疗联合光动力疗法的辐射抵抗性细胞，是否对临床标准化疗药物更为敏感，以及靶向细胞存活或DNA修复通路的小分子抑制剂能否提升受试疗法的疗效。RECA或X-PDT疗法方案的疗效将通过以下多项指标评估：检测癌细胞活力与诱导的细胞死亡类型、评估活性氧生成与线粒体活性、通过集落形成单位实验（colony-forming unit assay）检测辐射敏感性。本研究假说将在人源HNSCC与PCa小鼠模型中进行验证。在免疫健全的HNSCC与PCa小鼠模型（immunocompetent mouse models）中，我们将明确触发抗肿瘤免疫应答的潜力，以及免疫细胞在受试治疗方案中的作用。 本项目具有创新性，其旨在开发一种全新的肿瘤细胞放疗增敏方法，利用临床中已可用的具有光敏潜力的药物，可显著提升放疗抵抗性头颈部与前列腺肿瘤的放疗疗效。