Multiplexed time domain fluorescence tomography of tumor biomarkers during immunotherapy

摘要：使用程序性死亡1受体(PD-1)阻断的免疫治疗，无论是单独还是在 与现有疗法相结合，已被证明显著提高了许多患者的存活率 癌症，包括三阴性乳腺癌(TNBC)。然而，只有大约四分之一的人 患者对治疗有反应，通常是程序性死亡配体1(PD-L1)阳性的患者 肿瘤，而大多数人经历严重的药物相关副作用。可能的有效选择 免疫治疗的应答者受到以下事实的限制：活组织检查，目前的筛查标准 PD-L1表达，仅提供单个时间点生物标记物状态的快照，而它是 已知PD-L1的表达可以在治疗过程中动态变化。此外，肿瘤血管 “正常化”指标，如灌流、缺氧和血管生成可以动态变化 在治疗过程中，有可能成为治疗效果的早期指标。因此，有一种 迫切需要非侵入性成像技术，能够纵向量化分子和 治疗前和治疗中的生理预测肿瘤生物标志物。这种技术可以 有可能将无反应者从无效的治疗和危及生命的影响中拯救出来，还可以 促进对可提高存活率并被证明有效的新联合疗法的有力评估 一个更大的病人群体。我们使用时间域荧光成像的初步研究表明 帕金森病患者免疫受体靶向近红外探针的荧光寿命较长。 L1阳性肿瘤与正常组织中非特异性探针相比，从而显著改善 与基于荧光强度的成像相比，具有更高的灵敏度和特异度。此外，时间 区域成像允许同时检测和定量多个荧光团 光谱和寿命对比度(多路复用)，因此是成像多分子和 治疗反应的生理参数。这项提议的目标是将这些强大的 FLT验证层析FLT成像作为一种新的纵向多路扫描工具的益处 免疫治疗期间生物标志物的监测。我们将验证光学读数的准确性 用于纵向监测携带TNBC的小鼠的治疗反应 组织学。荧光成像的可行性以前已经被证明是表面的。 淋巴组织和乳房等器官。因此，Flt多路复接的有效性在 临床前模型是导致评估TD的有针对性的临床试验的根本步骤 TNBC患者非侵入性功能免疫治疗筛查技术。

Abstract: Immunotherapy using programmed death 1 receptor (PD-1) blockade, either alone or in combination with existing therapies, has been proven to significantly improve survival rates for many cancers, including triple negative breast cancer (TNBC). However, only about a quarter of the patients respond to treatment, typically those with programmed death ligand 1 (PD-L1)-positive tumors, while a majority experience serious drug related side effects. The efficient selection of likely responders to immunotherapy is limited by the fact that biopsy, the current screening standard for PD-L1 expression, provides only a snapshot of biomarker status at a single time point, while it is known that PD-L1 expression can dynamically change during therapy. Additionally, tumor vascular “normalization” indicators, such as perfusion, hypoxia and angiogenesis can dynamically change during treatment, potentially serving as early indicators of treatment efficacy. There is therefore an urgent need for non-invasive imaging techniques that can longitudinally quantify molecular and physiological predictive tumor biomarkers before and during treatment. Such techniques can potentially save non-responders from ineffective treatment and life-threatening effects and can also facilitate a robust evaluation of new combination therapies that improve survival and prove effective in a larger patient population. Our preliminary studies using time domain fluorescence imaging indicate that the fluorescence lifetime (FLT) of immune-receptor targeted near infrared probes is longer in PD- L1 positive tumors compared to non-specific probe in normal tissue, thereby dramatically improving sensitivity and specificity compared to fluorescence intensity-based imaging. Furthermore, time domain imaging allows the simultaneous detection and quantification of multiple fluorophores using spectral and lifetime contrast (multiplexing) and is therefore ideal for imaging multiple molecular and physiologic parameters of treatment response. The goal of this proposal is to translate these powerful benefits of FLT to validate tomographic FLT imaging as a new tool for multiplexed longitudinal monitoring of biomarkers during immunotherapy. We will validate the accuracy of the optical readouts for monitoring therapeutic response longitudinally in TNBC-bearing mice by comparison with histology. The feasibility of fluorescence imaging has previously been demonstrated for superficial lymph nodes and for organs such as the breast. Therefore, validation of FLT multiplexing in preclinical models is a fundamental step that will lead to targeted clinical trials to evaluate TD technology for non-invasive functional immunotherapy screening in TNBC patients.