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 al...