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.

The frozen tissue was cryopulverized and the protein was extracted into a 6M urea solution. Protein concentrations were determined by Micro BCA and the lysates were reduced, alkylated, and proteolytically digested using Lys-C and trypsin. After digestion, a mixture of SIS peptides was spiked into the individual samples at 75 or 200 fmol/sample of protein lysate, and the samples were desalted. Peptide immunoaffinity enrichment out of 500 μg of trypsin-digested lysate consisted of sequential overnight incubations of the cross-linked mAbs using a mixture of 1 µg of each of 55 antibodies followed by a mixture of 39 antibodies. Phosphopeptide enrichment by immobilized metal affinity chromatography (IMAC) out of 200 μg of trypsin-digested lysate was performed using Ni-NTA-agarose beads (Qiagen, Valencia, CA,) stripped with EDTA and incubated in a 10 mM FeCl3 solution to prepare Fe3+-NTA-agarose beads. A KingFisher platform was used to wash and elute the beads. For LC-MRM-MS analysis, 10 µL of a 26 µL eluate were injected on an ekspert nanoLC 425 with an NLC 400x AS autosampler coupled to a 5500 QTRAP mass spectrometer.

Melanoma-bearing PDX mice were subjected to total body irradiation. After one hour, tumors were harvested and quadrisected. Two parts of each tissue were snap-frozen in liquid nitrogen, and the remaining two parts were rapidly frozen in the quick-freeze prototypes for 1 hour before being transferred to an LN2 tank.