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志物开发来预测 治疗反应以及检测新疗法的潜在潜在药物靶点。经常出现的 实体瘤的局限性在于标准组织活检并不总是可行、安全或易于重复 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，无论 的来源。此外，随着免疫疗法的出现，重复采样变得更加重要 了解肿瘤和全身免疫反应，以更好地预测反应并预防耐药性。 因此，迫切需要开发组织活检的可靠且有效的替代方法。周边 血液的获取既简单又安全，并且在治疗前、治疗期间和治疗后更容易获取。周边 可以从标准全血中分离血液单核细胞 (PBMC)，然后进行分离和纯化 蛋白质、DNA 和 RNA 分析有可能作为组织抗癌反应的替代指标 治疗。然而，免疫功能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，对于 例如，新鲜样品的可重复活力对于包括细胞在内的功能反应非常重要 细胞毒性和趋化性。然而，新鲜处理和后续分析通常需要灵活的人员配置 由于患者样本采集时间不可预测，仪器的可用性始终保持不变。此外， 需要立即分析可能会妨碍批量样品的好处。这个假设的中心假设 建议优化 PBMC 处理将允许延迟且更全面、可重复 反映患者免疫和肿瘤状态的功能分析允许临床治疗决策 无需组织活检。 该假设将通过首先确定最佳收集、处理和存储来进行检验 最大限度地提高长期生存能力并维持完整的下游有意义的功能性免疫的条件 即使以延迟批处理方式执行分析。其次，我们将确定 PBMC 是否可重复 功能结果可作为肿瘤浸润免疫细胞功能和治疗效果的替代指标。 这种方法将使外周血生物样本的进步能够反映潜在的 肿瘤行为的机制以前被归结为侵入性组织活检。此外，我们还将有 建立了处理 PBMC 的条件，允许可重复地收集活细胞，从而维持 储存后的功能容量，可以通过不同的方法进行有意义的功能测定 设施。从该提案中获得的基础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，可以轻松获得患者样本 实时确定免疫疗法和其他抗癌疗法的治疗和临床反应。