Optimization of peripheral blood mononuclear cell (PBMC) processing for robust downstream functional immune cell analysis and correlation with therapeutic efficacy

人们越来越依赖于复杂的生物标志物的开发，以预测 治疗反应以及检测新疗法的潜在潜在药物靶点。经常 对于实体瘤的局限性在于标准组织活检并不总是可行的、安全的或容易重复的， 治疗最佳的样品采集、处理和最终验证对于任何生物标志物都至关重要， 的来源。此外，随着免疫疗法的出现，重复采样变得更加关键 了解肿瘤和全身免疫反应，以更好地预测反应和预防耐药性。 因此，迫切需要开发组织活检的可靠和有效的替代方案。外围 血液容易且安全地获得，并且在治疗之前、期间和之后更容易获得。外围 血液单核细胞（PBMC）可以从标准全血中分离，随后分离， 蛋白质、DNA和RNA的分析有可能作为组织对抗癌反应的替代物 疗法然而，免疫功能的分析更能反映全身和肿瘤的免疫反应， 使用PBMC的免疫疗法需要异常严格的处理技术。我们发现，由于 例如，新鲜样品的可再现活力对于功能反应，包括细胞反应， 细胞毒性和趋化性。然而，新鲜处理和随后的分析往往需要灵活的人员配置 以及由于患者样本收集的不可预测的时间而导致的恒定的仪器可用性。此外，委员会认为， 需要立即分析可能排除了快速取样的益处。这个问题的核心假设是 建议是，优化PBMC处理将允许延迟和更全面的，可重复的 反映患者免疫和肿瘤状态的功能分析，允许临床治疗决策 而不需要组织活检。 将通过首先确定最佳收集、处理和储存来检验假设 最大化长期生存力并维持完整的下游有意义的功能性免疫的条件 即使以延迟批次的方式进行分析。第二，我们将确定可重复的PBMC 功能结果作为肿瘤浸润免疫细胞功能和治疗功效的替代物。 这种方法将使外周血生物标本的进步，以反映潜在的 肿瘤行为的机制以前归入侵入性组织活检。此外，我们将有 建立了用于处理PBMC的条件，其允许可再现地收集维持 储存后的功能容量，从中可以通过不同的方法进行有意义的功能测定。 设施从本建议中获得的基本知识将有助于开发合适的 用于未来临床试验的相关PBMC分析允许容易获得的患者样本， 真实的确定对免疫疗法和其他抗癌疗法的治疗和临床反应。

There is an increasing dependence on sophisticated biomarker development to allow prediction of therapeutic response as well as detection of potential underlying drug targets for novel therapeutics. A frequent limitation for solid tumors is that standard tissue biopsies are not always feasible, safe or easily repeated during treatment. Optimal sample acquisition, processing, and final validation are critical for any biomarker, regardless of source. Moreover, with the advent of immunotherapy, repeated sampling has become even more critical to understand the tumor and systemic immune response to better predict response and prevent resistance. Accordingly, there is an urgent need to develop reliable and valid alternatives to tissue biopsies. Peripheral blood is easy and safe to obtain and is more readily obtainable before, during, and after treatment. Peripheral blood mononuclear cells (PBMCs) can be isolated from standard whole blood and subsequent isolation and analysis of protein, DNA and RNA has the potential to serve as a surrogate for tissue response to anti-cancer therapy. However, analysis of immune functions more reflective of the systemic and tumor immune response to immunotherapy, using PBMCs, requires unusually rigorous processing techniques. We have found, for example, that reproducible viability of fresh samples is important for functional responses including cellular cytotoxicity and chemotaxis. However, fresh processing with subsequent analysis often requires flexible staffing and constant instrumentation availability due the unpredictable timing of patient sample collection. Furthermore, requiring immediate analysis may preclude the benefits of batching samples. The central hypothesis of this proposal is that optimizing PBMC processing will allow for delayed and more comprehensive, reproducible functional analyses that reflect the patient immune and tumor status permitting clinical treatment decisions without the requirement of a tissue biopsy. The hypothesis will be tested by first determining the optimal collection, processing and storage conditions that maximize long-term viability and sustain intact downstream meaningful functional immune analyses even when performed in a delayed batch manner. Second, we will determine if the reproducible PBMC functional outcomes serve as a surrogates to tumor infiltrating immune cell function and therapeutic efficacy. This approach will allow the advancement of peripheral blood biospecimens to reflect underlying mechanisms of tumor behavior previously relegated to the invasive tissue biopsy. In addition, we will have established conditions for processing PBMCs that allow for reproducible collection of viable cells that maintain functional capacity upon storage, from which meaningful functional assays can be performed by different facilities. The fundamental knowledge obtained from this proposal will facilitate the development of suitable correlative PBMC analyses for future clinical trials allowing for an easily obtained patient specimen that can determine treatment and clinical responses in real time to immunotherapy and other anti-cancer therapy.