Preserving the phosphoproteome of clinical biopsies using a quick freeze collection device

The current gold standard for collecting and processing cancer biopsies is to flash freeze tumor samples in liquid nitrogen. However, in many clinical settings liquid nitrogen is not readily available, and neither the personnel nor the infrastructure is generally available to rapidly process the tumor samples. As a result, tumors are often subjected to prolonged ischemia and/or chemical fixatives, altering the phosphoproteome such that it may no longer reflect the true in vivo state of the tumor. There is a need for an economical, single-use device that can be stored at room temperature then activated at point of care to rapidly freeze the specimen. Our proof-of-concept quick-freeze prototype device focused on key requirements including cooling performance, device safety, minimizing use error, and the ability to ship via existing cold chain logistics. We have demonstrated that our device can cool a core sample below 0°C in less than 70 seconds, below -8°C in less than 150 seconds, and maintain that sample below 0°C for greater than 70 minutes. To demonstrate feasibility, the performance of our prototype was benchmarked against flash freezing in liquid nitrogen using melanoma-bearing PDX mice as a model system. After subjecting the mice to total body irradiation to elicit a phosphosignaling response in the DNA damage response pathway, tumors were harvested and quadrisected with two parts of the tumor snap frozen in liquid nitrogen, and the remaining two parts rapidly cooled in the prototype quick-freeze biospecimen containers for 1 hour. Phosphoproteins were profiled by LC-MS/MS. The prototype freeze device showed feasibility with bias within acceptable limits and slightly higher variability for a subset of phosphopeptides compared to flash freezing. The prototype device forms the framework for development of a commercial device that will improve tissue biopsy preservation for measurement of important phospho-signaling molecules.