Advancing Bladder Cancer Care by Imaging and Intravesical Immune Checkpoint Blockade

这项修订后的提案利用NCT04369560的初步发现来开发膀胱内成像和 膀胱癌免疫治疗途径(BCA)。而膀胱镜检查可以发现外生性肿瘤(≥2 mm in 直径)，现有成像方式的较差软组织分辨率使得它们不足以检测 与侵袭性癌相关的肿瘤侵袭、原位癌(CIS)和肿瘤血管。这 肿瘤可视化中的关键差距对高危BCA患者的早期识别和 最有可能从化疗和/或免疫治疗中受益的患者。虽然膀胱内免疫治疗 卡介苗(BCG)可阻止BCA进展和肌肉侵袭，仍有40%的患者表现出 卡介苗耐药肿瘤表达：程序性死亡(PD)L1、与PD1结合的PDL2配体 T细胞上的受体抑制抗肿瘤反应。而抗肿瘤反应则受到抑制 可注射抗体，继发于T细胞介导的免疫监视崩溃的不良反应 产生了对可注射抗体的膀胱内替代品的迫切需求，如Nivolumab。因此，我们将使用 致癌物N-丁基-N-4-羟丁基亚硝胺诱发的正交异性肿瘤PD-L1的过表达 (BBN)证明分子大小是滴注药物和染料渗透的关键决定因素 变成了癌症的焦点。因此，我们假设膀胱内对比增强磁共振成像 (MRI)在滴注加多布特洛尔(0.8 nm)和全氟十氢林乳剂(&gt；150 nm)后，利用 Gadobutrol向肿瘤的渗透和磁性惰性、全氟十氢化合物的大小受限扩散 增强癌灶图像对比度，实现肿瘤进展和肿瘤虚拟监测 小分子(640道尔顿)BMS-1166、PD-1/PD-L1膀胱内免疫治疗后的消退 抑制剂(IC50为1.4 nM)。我们的假设将在这些环环相扣的具体目标中得到检验：1)评估 膀胱内增强MRI监测BBN诱导的肿瘤进展的标准效度。2)至 评价膀胱内增强MRI监测免疫治疗的判别效度 BBN肿瘤消退。在水中随意喂食0.05%的BBN长达12周，我们会引发肿瘤 免疫活性18-24周龄B6D2F1小鼠正常和癌灶的定量T1弛豫测量 和肿瘤血管系统在不同的时间点监测BBN肿瘤的进展，后来被整体证实 移植物膀胱组织病理学和血管生成标记物的表达(目标1)。监督治疗情况 介导性肿瘤消退(AIM 2)，饲喂BBN 10周的小鼠将随机接受单一的 0.1ml尼伏单抗或卡介苗或不溶于水的BMS-1166滴入神经鞘氨醇体内 或50%DMSO，6周后行MRI检查，以评估治疗对肿瘤大小和 伴生的血管系统。因此，我们将使MRI能够对CIS、肌肉侵袭和肿瘤血管系统进行成像 以及开发一种新的膀胱内免疫治疗方案。

This revised proposal leverages the preliminary findings of NCT04369560 to develop intravesical imaging and immunotherapy approach for bladder cancer (BCa). While cystoscopy can detect exophytic tumors (≥2mm in diameter), the poor soft tissue resolution of the available imaging modalities renders them inadequate to detect tumor invasion, carcinoma-in-situ (CIS) and the tumor vasculature associated with aggressive cancer. This critical gap in tumor visualization adversely impacts the early identification of high risk BCa patients and patients most likely to benefit from chemotherapy and/or immunotherapy. Although intravesical immunotherapy of Bacillus Calmette–Guérin (BCG) halts BCa progression and muscle invasion, still 40% of patients exhibit BCG-resistant tumor with the expression of: programmed death (PD)L1, PDL2 ligands for engaging with PD1 receptor on T cells to suppress the anti-tumor response. While the anti-tumor response is inhibited by injectable antibodies, adverse effects secondary to the breakdown of T-cell mediated immune surveillance creates a dire need for intravesical alternatives to injectable antibodies like Nivolumab. Hence, we will use the overexpression of PD-L1 by orthotropic tumor provoked by carcinogen, N-butyl-N-4-hydroxybutyl nitrosamine (BBN) to demonstrate that molecular size is the key determinant for the permeation of instilled drugs and dyes into cancer foci. Accordingly, we hypothesize that intravesical contrast enhanced magnetic resonance imaging (MRI) after the instillation of Gadobutrol (0.8nm) mixed with Perfluorodecalin emulsion (>150nm) leverages the tumoritropic infiltration of Gadobutrol and the size-restricted diffusion of magnetically inert, Perfluorodecalin to enhance the image contrast of cancer foci for accomplishing virtual monitoring of tumor progression and tumor regression following intravesical immunotherapy of BMS-1166, a small molecule (640 Daltons), PD-1/PD-L1 inhibitor (IC50 of 1.4 nM). Our hypotheses will be tested in these interlocking Specific Aims: 1) To assess the criterion validity of intravesical contrast-enhanced MRI for monitoring BBN induced tumor progression. 2) To assess the discriminant validity of intravesical contrast-enhanced MRI for monitoring immunotherapy mediated BBN tumor regression. By ad libitum feeding of 0.05% BBN in water for up to 12 weeks, we will provoke tumor in immunocompetent 18-24 weeks old B6D2F1 mice for quantitative T1 relaxometry of normal and cancer foci and tumor vasculature at different time points to monitor BBN tumor progression, later confirmed by whole mount bladder histopathology and the expression of angiogenesis markers (Aim 1). To monitor the treatment mediated tumor regression (Aim 2), mice fed BBN for 10 weeks will be randomized to receive either a single 0.1mL instillation at 3mg/mL of Nivolumab or BCG or water-insoluble BMS-1166 entrapped in Sphingosomes or 50% DMSO followed by MRI, 6 weeks later to assess the effect of treatment on tumor size and the associated vasculature. Thus, we will enable MRI for imaging CIS, muscle invasion and tumor vasculature as well as develop a novel intravesical immunotherapeutic option.