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

优化外周血单核细胞 (PBMC) 处理，以实现强大的下游功能性免疫细胞分析以及与治疗效果的相关性

• 批准号: 10569111
• 负责人: Kelsey Dillehay McKillip
• 金额: $ 35.04万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569111
• 关键词: Activities of Daily Living; Aftercare; Biological Assay; Biological Markers; Biopsy; Blood specimen; Cell Separation; Cell Survival; Cell physiology; Cell-Mediated Cytolysis; Cells; Cellular Assay; Chemotaxis; Clinical; Clinical Treatment; Clinical Trials; Collaborations; Collection; Complement; DNA; Dependence; Detection; Development; Drug Targeting; Flow Cytometry; Future; Grant; Hematologic Neoplasms; Histology; Immune; Immune response; Immunohistochemistry; Immunotherapy; Infiltration; Knowledge; Malignant Neoplasms; Methods; Mission; Natural Killer Cells; Ohio; Pathway interactions; Patients; Peripheral; Peripheral Blood Mononuclear Cell; Prediction of Response to Therapy; Process; Protein Analysis; Protocols documentation; RNA; Reproducibility; Reproducibility of Results; Resistance; Safety; Sampling; Science; Site; Solid Neoplasm; Source; Specimen; Stress; Techniques; Testing; Therapeutic; Time; Tissues; Treatment Efficacy; Tube; Tumor Tissue; Tumor-infiltrating immune cells; Universities; Validation; Whole Blood; anticancer treatment; biomarker development; blood-based biomarker; cancer therapy; clinical decision-making; cytokine; cytotoxic; design; flexibility; functional outcomes; head and neck cancer patient; immune function; improved; individualized medicine; instrumentation; new therapeutic target; novel; novel therapeutics; outcome prediction; peripheral blood; predicting response; prevent; response; sample collection; survival outcome; treatment choice; treatment response; tumor; tumor behavior; tumor microenvironment; tumor-immune system interactions

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.

人们越来越依赖复杂的生物标记物的开发来预测 新疗法的治疗反应以及潜在潜在药物靶点的检测。常客 实体瘤的局限性在于，标准的组织活检并不总是可行、安全或容易重复的 治疗。无论如何，最佳的样本获取、处理和最终验证对任何生物标志物都至关重要 来源的问题。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，迫切需要开发可靠和有效的替代组织活检的方法。外围 血液是容易和安全的，而且在治疗前、治疗中和治疗后都更容易获得。外围 血液单个核细胞(PBMC)可以从标准全血和随后的分离和 蛋白质、DNA和RNA的分析有可能作为组织抗癌反应的替代物 心理治疗。然而，免疫功能的分析更能反映全身和肿瘤的免疫反应 使用外周血单核细胞的免疫疗法需要异常严格的处理技术。我们已经发现，对于 例如，新鲜样本的可重复性对于包括细胞在内的功能反应是重要的 细胞毒性和趋化性。然而，后续分析的新处理通常需要灵活的人员配置 由于患者样本采集的时间不可预测，仪器持续可用。此外， 要求立即进行分析可能会排除批量样品的好处。这一点的中心假设是 建议是，优化PBMC处理将允许延迟，更全面，可重复性 反映患者免疫和肿瘤状态的功能分析，可用于临床治疗决策 而不需要组织活检。 首先将通过确定最佳的收集、处理和存储来检验这一假设 使长期生存能力最大化并维持完整的下游有意义的功能性免疫的条件 即使以延迟批处理的方式执行分析。其次，我们将确定可复制的PBMC 功能结果可作为肿瘤浸润性免疫细胞功能和治疗效果的替代指标。 这种方法将允许外周血液生物标本的进步以反映潜在的 肿瘤行为的机制以前归类于侵袭性组织活检。此外，我们还将拥有 建立了处理PBMC的条件，允许可重复收集保持 存储时的功能容量，从中可以执行有意义的功能分析，由不同的 设施。从这项建议中获得的基本知识将有助于开发适用的 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 实时确定对免疫治疗和其他抗癌治疗的治疗和临床反应。