Designing a transcriptome-based, targeted theranostic platform for prostate cancer

摘要 缺乏经过验证的分子特征能够清楚地区分前列腺癌的侵袭性表型，以及 缺乏合适的中间终点使得该肿瘤难以合理地管理。我们有 开创并开发了一种体内筛选系统，其中能够归巢的肽靶向颗粒 在静脉内给药后从噬菌体展示随机文库中回收。中 对具有几种生物学属性的已建立的患者源性异种移植物（PDX）进行无偏筛选 让人想起人类转移性前列腺癌，我们首先分离并验证了肿瘤归巢肽 靶向细胞表面相关葡萄糖调节蛋白-78kD（GRP 78）;我们还筛选了一名患者， 直接分离和验证靶向白细胞介素-11受体（IL 11 R）的配体。我们接下来插入顺- 基因元件从腺相关病毒（AAV）噬菌体基因组内，并建立了杂交 配体导向的AAV/噬菌体（AAVP）颗粒能够全身性肿瘤靶向递送和分子遗传学治疗。 前列腺癌临床前模型中报告基因的成像。我们假设， 前列腺癌中上调的转基因应该能够使转录驱动的自杀基因表达， 肿瘤成像与串联生长抑制。我们小组的工作（其中包括）表明， GRP 78和IL 11 R的过度表达与疾病进展有关。因此，这些基因的启动子 将在新的AAVP结构中进行评价。在目标1中，我们将验证GRP 78和IL 11 R，此外， 通过前列腺癌患者来源的样品的转录组分析鉴定的其它基因的启动子。 这些启动子将被克隆到报告质粒中，以初步评估它们相对于标准启动子的特性。 CMV启动子在前列腺癌细胞和三维患者源性成骨前列腺癌培养物中的表达 体外在目标2中，将在动物模型中评价构建体。我们的策略是通过静脉注射 注射GRP 78靶向AAVP，其经工程改造以递送单纯疱疹病毒胸苷激酶（HSVtk） 转基因。启动子驱动的TK表达将通过PET/CT用[18 F]-FEAU成像，并通过 qPCR。更昔洛韦治疗后的肿瘤生长抑制将通过成像在体内进行评估，并且肿瘤生长抑制将在体内进行评估。 将通过免疫组织化学评价切片。转录谱的整合，前列腺 在单一平台内的癌细胞靶向和分子遗传成像将可能允许 监测病理学上难以区分但生物学上多样性的疾病进展和对治疗的反应 不同疾病结局的患者队列。鉴于先前的工作机构已经建立了GRP 78 和IL 11 R作为人类前列腺癌的真正分子靶点，我们的方法学方法和 研究结果具有机械和翻译意义。

ABSTRACT The lack of validated molecular profiles clearly distinguishing aggressive phenotypes of prostate cancer, and the absence of suitable intermediate endpoints make this tumor difficult to manage rationally. We have pioneered and developed an in vivo screening system in which peptide-targeted particles capable of homing to tumors are recovered from a phage display random library following intravenous administration. In an unbiased screening of an established patient-derived xenograft (PDX) with several biological attributes reminiscent of human metastatic prostate cancer, we first isolated and validated tumor-homing peptides targeting the cell surface-associated glucose-regulated protein-78 kD (GRP78); we also screened a patient directly to isolate and validate ligands targeting the interleukin-11 receptor (IL11R). We next inserted cis- genetic elements from adeno-associated virus (AAV) within the phage genome and established that hybrid ligand-directed AAV/phage (AAVP) particles enable systemic tumor targeted delivery and molecular-genetic imaging of reporter transgene in preclinical models of prostate cancer. We hypothesize that promoters of upregulated transgenes in prostate cancer should enable transcription-driven suicide gene expression for tumor imaging with in tandem growth suppression. Work from our group (among others) shows that overexpression of GRP78 and IL11R is related to disease progression. As such, promoters for these genes will be evaluated in new AAVP constructs. In Aim 1, we will validate GRP78 and IL11R, in addition to promoters of other genes identified by transcriptome analysis of prostate cancer patient-derived samples. These promoters will be cloned into reporter plasmids to initially assess their properties relative to a standard CMV promoter in prostate cancer cells and 3-dimensional patient-derived osteogenic prostate cancer cultures in vitro. In Aim 2, the constructs will be evaluated in animal models. Our strategy relies on intravenously injected GRP78-targeting AAVP engineered to deliver a Herpes Simplex Virus thymidine kinase (HSVtk) transgene. Promoter-driven TK expression will be imaged with [18F]-FEAU by PET/CT and quantified by qPCR. Tumor growth inhibition after ganciclovir treatment will be assessed in vivo by imaging, and tumor sections will be evaluated by immunohistochemistry. The integration of transcriptional profiling, prostate cancer cell targeting and molecular-genetic imaging within a single platform will potentially allow for the monitoring of progression and response to therapy in pathologically indistinguishable, yet biologically diverse patient cohorts with different disease outcomes. Given that the previous body of work has established GRP78 and IL11R as bona fide molecular targets in human prostate cancer, our methodological approach and findings bear mechanistic and translational significance.