Project 1: 5FU and Vitamin D as Neoadjuvants for Photodynamic Priming to Enhance Skin Cancer Therapy

摘要-项目1 项目1将探讨如何光动力疗法（PDT）可以用于免疫启动在新的 皮肤癌的综合治疗非黑素瘤皮肤癌（NMSC），包括鳞状细胞癌 癌（SCC）和基底细胞癌（BCC）是所有人类癌症中最常见的，代表了一种恶性肿瘤。 显著的医疗保健负担（> 300万美国病例/年），某些患者亚群的致死率增加， 例如器官移植接受者我们以前表明，氨基乙酰丙酸（ALA）为基础的PDT可以有效地 对于NMSC患者，5-氟尿嘧啶（5 FU）和维生素D（VitD）进一步改善治疗结果;但 大的和晚期的NMSC的肿瘤清除率仍然很差。为改善这情况，我们建议 利用光动力引发（PDP）的概念，PDT的自然结果。PDT触发免疫原性 细胞死亡，增强肿瘤浸润淋巴细胞（TIL）的流入，并诱导适应性抗肿瘤免疫 免疫检查点抑制可以通过增强免疫应答，使肿瘤微环境对免疫检查点抑制的作用敏感。我们 初步数据显示低强度PDT方案可以清除鼠SCC模型中的肿瘤， 伴随着损伤相关分子模式（DAMP）的诱导和中性粒细胞的募集， 巨噬细胞和TIL，沿着免疫检查点分子如PD-1的表达改变。这些 这些发现为使用PDT和免疫检查点抑制剂（ICI）的进一步组合方法奠定了基础。在 目的1（临床前），我们将在小鼠SCC和BCC模型中评估对各种抗肿瘤药物的治疗反应。 PDT、5 FU、VitD和ICI试剂的组合，并测量TIL募集、检查点 分子表达和肿瘤清除。在目标2（临床研究）中，我们将确定 BCC和SCC患者PDT后的免疫反应。肿瘤将接受PDT（由核心 C剂量测定法以测量光敏剂水平）;然后在1至14天后，手术切除肿瘤， 使用组织学方法分析检查点分子表达和肿瘤内免疫细胞浸润。 免疫评分（IS;项目3）。将合作测量循环血液中的全身性T细胞活化 项目2。这些研究的另一个分支将测试NMSC肿瘤的5 FU或VitD预处理对肿瘤生长的影响。 抗肿瘤免疫反应的强度。目标3将以探索性研究为特色，以验证IS 在体外测量人SCC肿瘤模型中TIL募集的方法。与项目3合作 和核心B，3D患者源性肿瘤免疫类器官（PDIO），由分层SCC培养物组成 与PBMC（淋巴细胞）混合后，将在PDT后使用 核心B开发的尖端高光谱光学细胞术技术，可以成像多种免疫 标志同时相关性和影响：总体而言，该项目将通过提供 一种新方法的基础，结合光动力学引发，5 FU，VitD和ICI免疫疗法，以改善 NMSC疑难和晚期病例的治疗结果。

ABSTRACT – Project 1 Project 1 will explore how photodynamic therapy (PDT) can be used for immunological priming in new combination treatments for skin cancer. Nonmelanoma skin cancers (NMSC), comprising squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), are the most common of all human cancers, representing a significant healthcare burden (>3 million U.S. cases/year), with increased lethality for certain patient subsets, e.g. organ transplant recipients. We showed previously that aminolevulinate (ALA)-based PDT can be effective for NMSC patients, and that 5-fluorouracil (5FU) and Vitamin D (VitD) further improve treatment outcomes; yet tumor clearance rates remain poor for large and advanced NMSC. To ameliorate this situation, we propose to exploit the concept of photodynamic priming (PDP), a natural consequence of PDT. PDT triggers immunogenic cell death, enhances influx of tumor infiltrating lymphocytes (TILs), and induces an adaptive anti-tumor immune response, sensitizing the tumor microenvironment to the effects of immune checkpoint inhibition. Our preliminary data show that a low-intensity PDT regimen can clear tumors in a murine SCC model, accompanied by induction of damage-associated molecular patterns (DAMPs) and recruitment of neutrophils, macrophages and TILs, along with altered expression of immune checkpoint molecules such as PD-1. These findings set the stage for a further combination approach using PDT and immune checkpoint inhibitors (ICI). In Aim 1 (preclinical) we will evaluate in murine SCC and BCC models the therapeutic response to various combinations of PDT, 5FU, VitD, and ICI agents, and measure specific timing of TIL recruitment, checkpoint molecule expression, and tumor clearance. In Aim 2 (clinical studies) we will determine the nature and timing of immune responses in BCC and SCC patients, post-PDT. Tumors will be subjected to PDT (guided by Core C dosimetry to measure photosensitizer levels); then after 1-to-14 days the tumor will be surgically excised and analyzed for checkpoint molecule expression and intratumoral immune cell infiltration using the histological immunoscore (IS; Project 3). Systemic T-cell activation in circulating blood will be measured in collaboration with Project 2. Another arm of these studies will test effects of 5FU or VitD pretreatment of NMSC tumors upon the magnitude of anti-tumoral immune responses. Aim 3 will feature exploratory studies to validate the IS approach for measuring TIL recruitment in human SCC tumor models in vitro. In collaboration with Project 3 and Core B, 3D patient-derived tumor-immune organoids (PDIOs), consisting of stratified SCC cultures admixed with PBMCs (lymphocytes) will be tested for differential immune cell activation following PDT using cutting-edge hyperspectral optical cytometry techniques developed in Core B that can image multiple immune markers simultaneously. Relevance and Impact: Overall, this Project will benefit patients by providing the basis for a new approach that combines photodynamic priming, 5FU, VitD, and ICI immunotherapy to improve treatment outcomes for difficult and advanced cases of NMSC.