Identification of serum protein biomarkers by profiling N-glycoproteomes of patient-derived xenografts of neuroendocrine prostate cancer

项目总结 神经内分泌前列腺癌(NEPC)是前列腺癌(PCa)的一种致命性变种，可导致前列腺癌 在17-30%以前接受标准护理雄激素剥夺疗法(ADT)治疗的患者中 前列腺癌(AC)作为一种耐药机制。血清生物标记物具有高敏感性和 临床医生迫切需要非侵入性检测NEPC的特异性，以便选择合适的 及时进行一线系统治疗，选择患者进行临床试验，并监测治疗情况 需要颠倒NEPC状态的响应。一种新的策略，可以快速识别人类- 小鼠血清背景中独特的蛋白质，从而克服了与蛋白质组学相关的限制- 基于生物标志物的发现，已成功地用于识别卵巢中的血清糖蛋白生物标志物 癌症患者来源的异种移植(PDX)。我们假设反映人类前列腺癌的PCa PDX肿瘤 表达在荷瘤小鼠血清中容易识别的独特的肿瘤相关糖蛋白 使用质谱学。此外，不同的血清糖蛋白及其水平与不同的 PCA PDX的亚型，即纯AC、AC与NEPC混合，以及纯NEPC。最后，这些子类型 特异的生物标志物可用于鉴别PCa患者临床标本中的NEPC。 在目标1中，我们将通过以下方法鉴定与PCa PDX肿瘤亚型相关的新的血清糖蛋白 PDX血清糖蛋白组学分析。具体来说，PDX肿瘤将使用冷冻保存的组织产生 用肾下组织学方法从具有纯AC、AC与NEPC混合和纯NEPC组织学的公共可用PDXs系中获得 移植到免疫缺陷小鼠体内。将进行定量蛋白质组学以产生糖蛋白质组 将以生物信息学的方式选择PDX血清和人类特有蛋白的图谱。在目标2中，血清蛋白 在含有NEPC成分的样品中发现，但在纯AC样品中没有表达，将被选中。前五名 为其成功开发靶向分析的候选NEPC特异性生物标记物将在 来自PDX小鼠和具有经病理证实的NEPC成分的患者的血清。敏感度和 这些生物标记物的特异性将使用经病理证实的AC和 以良性前列腺增生症为真阴性对照。 我们的方法将导致生物标志物的快速开发，用于临床，以减少 病态活组织检查和对患者进行适当的分流，以适应他们的疾病状态，这反过来又将 提高生活质量和生存能力。此外，这些生物标志物可用于监测患者对 护理标准疗法和选定的患者进行临床试验，测试未来开发的新疗法 专门针对NEPC的。此外，我们鉴定的亚型特异性糖蛋白可以作为 为未来的成像战略或靶向治疗奠定基础。最后，NEPC的糖蛋白组会提供一种 为更好地理解NEPC发展机制提供了宝贵的资源。

PROJECT SUMMARY Neuroendocrine prostate cancer (NEPC) is a lethal variant of prostate cancer (PCa) that may arise de novo or in 17-30% of patients previously treated with standard of care androgen deprivation therapy (ADT) for prostate adenocarcinoma (AC) as a mechanism of resistance. Serum biomarkers with high sensitivity and specificity for non-invasive detection for NEPC are urgently needed to enable clinicians to select the proper next-line systemic therapy in a timely manner, select of patients for clinical trials, and monitor treatment responses that entail reversing the NEPC state. A novel strategy that allows quick identification of ‘‘human- unique’’ proteins in a mouse serum background, thereby overcoming limitations associated with proteomics- based biomarker discovery, has been used successfully to identify serum glycoprotein biomarkers in ovarian cancer patient-derived xenografts (PDX). We hypothesize that PCa PDX tumors that reflect human PCa express unique tumor-associated glycoproteins that can be readily identified in sera of tumor-bearing mice using mass spectrometry. Moreover, different serum glycoproteins and their levels are associated with different subtypes of PCa PDXs, namely, pure AC, AC mixed with NEPC, and pure NEPC. Finally, these subtype specific biomarkers can be used to identify NEPC in clinical samples from PCa patients. In Aim 1, we will identify novel serum glycoproteins associated with subtypes of PCa PDX tumors by glycoproteomic analyses of PDX sera. Specifically, PDX tumors will be generated using cryopreserved tissues from publically available PDXs lines with pure AC, AC mixed with NEPC and pure NEPC histology by subrenal implantation into immunodeficient mice. Quantitative proteomics will be performed to generate glycoproteomic profiles of PDX sera and human-specific proteins will be selected bioinformatically. In Aim 2, serum proteins found in samples with a NEPC component but not expressed in pure AC samples will be selected. The top 5 candidate NEPC-specific biomarkers for which a targeted assay is developed successfully will be validated in sera from PDX mice and patients with pathologically confirmed NEPC component. The sensitivity and specificity of these biomarkers will be assessed using sera from patients with pathologically confirmed AC and benign prostatic hyperplasia as true negative control. Our approach would lead to biomarkers that could be developed quickly for use in the clinic to reduce morbid biopsies and appropriately triage patients to therapies tailored to their disease state, which in turn, will improve quality of life and survival. In addition, these biomarkers could be used to monitor patient response to standard-of-care therapies and select patients for clinical trials testing novel therapies developed in the future that target NEPC specifically. Moreover, the subtype-specific glycoproteins we identified could serve as the basis for future imaging strategies or targeted therapies. Finally, the glycoproteomes of NEPC will provide a valuable resource for better understanding the mechanisms of NEPC development.